Compositions and methods for enhancing drug delivery across and into epithelial tissues

ABSTRACT

This invention provides compositions and methods for enhancing delivery of drugs and other agents across epithelial tissues, including the skin, gastrointestinal tract, pulmonary epithelium, and the like. The compositions and methods are also useful for delivery across endothelial tissues, including the blood brain barrier. The compositions and methods employ a delivery enhancing transporter that has sufficient guanidino or amidino sidechain moieties to enhance delivery of a compound conjugated to the reagent across one or more layers of the tissue, compared to the non-conjugated compound. The delivery-enhancing polymers include, for example, poly-arginine molecules that are preferably between about 6 and 25 residues in length.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplication No. 60/150,510, filed Aug. 24, 1999. This application isrelated to U.S. patent application No. ________, filed on even dateherewith as TTC Attorney Docket No. 019801-000220US. Both of theseapplications are incorporated herein by reference for all purposes

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention pertains to the field of compositions and methodsthat enhance the delivery of drugs and other compounds across the dermaland epithelial membranes, including, for example, skin, thegastrointestinal epithelium and the bronchial epithelium.

[0004] 2. Background

[0005] Transdermal or transmucosal drug delivery is an attractive routeof drug delivery for several reasons. Gastrointestinal drug degradationand the hepatic first-pass effect are avoided. In addition, transdermaland transmucosal drug delivery is well-suited to controlled, sustaineddelivery (see, e.g., Elias, In Percutaneous Absorption:Mechanisms-Methodology-Drug Delivery, Bronaugh & Maibach, Eds., pp 1-12,Marcel Dekker, New York, 1989.). For many applications, traditionalmethods of administering drugs are not optimal because of the very largeinitial concentration of the drug. Transdermal delivery could allow amore uniform, slower rate of delivery of a drug. Moreover, patientcompliance is encouraged because such delivery methods are easy to use,comfortable, convenient and non-invasive.

[0006] These advantages of transdernal and transmucosal delivery havenot led to many clinical applications because of the low permeability ofepithelial membranes, the skin in particular, to drugs. The difficultiesin delivering drugs across the skin result from the barrier property ofskin. Skin is a structurally complex thick membrane that represents thebody's border to the external hostile environment. The skin is composedof the epidermis, the dermis, the hypodermis, and the adenexalstructures (epidermal appendages). The epidermis, the outermostepithelial tissue of the skin, is itself composed of several layers—thestratum corneum, the stratum granulosum, the stratum spinosum, and thestratum basale.

[0007] Compounds that move from the environment into and through intactskin must first penetrate the stratum corneum, the outermost layer ofskin, which is compact and highly keratinized. The stratum corneum iscomposed of several layers of keratin-filled skin cells that are tightlybound together by a “glue” composed of cholesterol and fatty acids. Thethickness of the stratum corneum varies depending upon body location. Itis the presence of stratum corneum that results in the impermeability ofthe skin to pharmaceutical agents. The stratum corneum is formednaturally by cells migrating from the basal layer toward the skinsurface where they are eventually sloughed off. As the cells progresstoward the surface, they become progressively more dehydrated andkeratinized. The penetration across the stratum corneum layer isgenerally the rate-limiting step of drug permeation across skin. See,e.g., Flynn, G. L., In Percutaneous Absorption:Mechanisms-Methodology-Drug Delivery, supra. at pages 27-53.

[0008] After penetration through the stratum corneum layer, systemicallyacting drug molecules then must pass into and through the epidermis, thedermis, and finally through the capillary walls of the bloodstream. Theepidermis, which lies under the stratum corneum, is composed of threelayers. The outermost of these layers is the stratum granulosum, whichlies adjacent to the stratum corneum, is composed of cells that aredifferentiated from basal cells and keratinocytes, which make up theunderlying layers having acquired additional keratin and a moreflattened shape. The cells of this layer of the epidermis, which containgranules that are composed largely of the protein filaggrin. Thisprotein is believed to bind to the keratin filaments to form the keratincomplex. The cells also synthesize lipids that function as a “cement” tohold the cells together. The epidermis, in particular the stratumgranulosum, contains enzymes such as aminopeptidases.

[0009] The next-outermost layer of the epidermis is the stratumspinosum, the principal cells of which are keratinocytes, which arederived from basal cells that comprise the basal cell layer. Langerhanscells, which are also found in the stratum spinosum, areantigen-presenting cells and thus are involved in the mounting of animmune response against antigens that pass into the skin. The cells ofthis layer are generally involved in contact sensitivity dermatitis.

[0010] The innermost epidermal layer is the stratum basale, or basalcell layer, which consists of one cell layer of cuboidal cells that areattached by hemi-desmosomes to a thin basement membrane which separatesthe basal cell layer from the underlying dermis. The cells of the basallayer are relatively undifferentiated, proliferating cells that serve asa progenitor of the outer layers of the epidermis. The basal cell layerincludes, in addition to the basal cells, melanocytes.

[0011] The dermis is found under the epidermis, which is separated fromthe dermis by a basement membrane that consists of interlocking reteridges and dermal papillae. The dermis itself is composed of two layers,the papillary dermis and the reticular dermis. The dermis consists offibroblasts, histiocytes, endothelial cells, perivascular macrophagesand dendritic cells, mast cells, smooth muscle cells, and cells ofperipheral nerves and their end-organ receptors. The dermis alsoincludes fibrous materials such as collagen and reticulin, as well as aground substance (principally glycosaminoglycans, including hyaluronicacid, chondroitin sulfate, and dermatan sulfate).

[0012] Several methods have been proposed to enhance transdermaltransport of drugs. For example, chemical enhancers (Bumette, R. R. InDevelopmental Issues and Research Initiatives; Hadgraft J., Ed., MarcelDekker: 1989; pp. 247-288), iontophoresis, and others have been used.However, in spite of the more than thirty years of research that hasgone into delivery of drugs across the skin in particular, fewer than adozen drugs are now available for transdermal administration in, forexample, skin patches.

[0013] Transport of drugs and other molecules across the blood-brainbarrier is also problematic. The brain capillaries that make up theblood-brain barrier are composed of endothelial cells that form tightjunctions between themselves (Goldstein et al., Scientific American255:74-83 (1986); Pardridge, W. M., Endocrin. Rev. 7: 314-330 (1986)).The endothelial cells and the tight intercellular junctions that jointhe cells form a barrier against the passive movement of many moleculesfrom the blood to the brain. The endothelial cells of the blood-brainbarrier have few pinocytotic vesicles, which in other tissues can allowsomewhat unselective transport across the capillary wall. Nor is theblood-brain barrier interrupted by continuous gaps or channels that runthrough the cells, thus allowing for unrestrained passage of drugs andother molecules.

[0014] Thus, a need exists for improved reagents and methods forenhancing delivery of compounds, including drugs, across epithelialtissues and endothelial tissues such as the skin and the blood-brainbarrier. The present invention fulfills this and other needs.

SUMMARY OF THE INVENTION

[0015] The present invention provides methods for enhancing delivery ofa compound into and across one or more layers of an animal epithelial orendothelial tissue. The methods involve contacting tissue with aconjugate that includes the compound and a delivery-enhancingtransporter. The delivery-enhancing transporters, which are alsoprovided by the invention, have sufficient guanidino or amidino moietiesto increase delivery of the conjugate into and across one or more intactepithelial or endothelial tissue layers compared to delivery of thecompound in the absence of the delivery-enhancing transporter.Typically, the delivery-enhancing transporters have from 6 to 25guanidino or amidino moieties, and more preferably between 7 and 15guanidino moieties.

[0016] The delivery-enhancing transporters and methods of the inventionare useful for delivering drugs, diagnostic agents, and other compoundsof interest across epithelial tissues such as the skin and mucousmembranes. Delivery across the blood-brain barrier is also enhanced bythe conjugates and methods of the invention. The methods andcompositions of the invention can be used not only to deliver thecompounds to the particular site of administration, but also providesystemic delivery.

[0017] In some embodiments, the delivery-enhancing transporter comprises7-15 arginine residues or analogs of arginine. The delivery-enhancingtransporter can have at least one arginine that is a D-arginine and insome embodiments, all arginines are D-arginine. The delivery-enhancingtransporter can consist essentially of 5 to 50 amino acids, at least 50percent of which are arginine. In some embodiments, at least 70% of theamino acids are arginines or arginine analogs. In some embodiments, thedelivery-enhancing transporter comprises at least 5 contiguous argininesor arginine analogs.

[0018] The compound to be delivered can be connected to the deliveryenhancing transporter by a linker. In some embodiments, the linker is areleasable linker which releases the compound, in biologically activeform, from the delivery-enhancing transporter after the compound haspassed into and through one or more layers of the epithelial and/orendothelial tissue. In some embodiments, the compound is released fromthe linker by solvent-mediated cleavage. The conjugate is, in someembodiments, substantially stable at acidic pH but the compound issubstantially released from the delivery-enhancing transporter atphysiological pH. In some embodiments, the half-life of the conjugate isbetween 5 minutes and 24 hours upon contact with the skin or otherepithelial or endothelial tissue. For example, the half-life can bebetween 30 minutes and 2 hours upon contact with the skin or otherepithelial or endothelial tissue.

[0019] Examples of conjugate structures of the invention include thosehaving structures such as 3, 4, or 5, as follows:

[0020] where R₁—X comprises the compound; X is a functional group on thecompound to which the linker is attached; Y is N or C; R₂ is hydrogen,alkyl, aryl, acyl, or allyl; R₃ comprises the delivery-enhancingtransporter; R₄ is substituted or unsubstituted S, O, N or C; R₅ is OH,SH or NHR₆; R₆ is hydrogen, alkyl, aryl, acyl or allyl; k and m are eachindependently selected from 1 and 2; and n is 1 to 10. Preferably, X isselected from the group consisting of N, O, S, and CR₇R₈, wherein R₇ andR₈ are each independently selected from the group consisting of H andalkyl. In some embodiments, R₄ is S; R₅ is NHR₆; and R₆ is hydrogen,methyl, allyl, butyl or phenyl. In some embodiments, R₂ is benzyl; k, m,and n are each 1, and X is O. In some embodiments, the conjugatecomprises structure 3 and R₂ is selected to obtain a conjugate half-lifeof between 5 minutes and 24 hours. In some embodiments, R₂ is selectedto obtain a conjugate half-life of between 5 minutes and 24 hours. Insome embodiments, the conjugate comprises structure 4; R₄ is S; R₅ isNHR₆; and R₆ is hydrogen, methyl, allyl, butyl or phenyl. In someembodiments, the conjugate comprises structure 4; R₅ is NHR₆; R₆ ishydrogen, methyl, allyl, butyl or phenyl; and k and m are each 1. Oneexample of a conjugate is:

[0021] where Ph is phenyl.

[0022] The invention also provides conjugates in which the release ofthe linker from the biological agent involves a first, rate-limitingintramolecular reaction, followed by a faster intramolecular reactionthat results in release of the linker. The rate-limiting reaction can,by appropriate choice of substituents of the linker, be made to bestable at a pH that is higher or lower than physiological pH. However,once the conjugate has passed into and across one or more layers of anepithelial or endothelial tissue, the linker will be cleaved from theagent. An example of a compound that has this type of linker isstructure 6, as follows:

[0023] wherein R₁—X comprises the compound to be delivered across one ormore layers of an epithelial and/or endothelial tissue; X is afunctional group on the compound to which the linker is attached; Ar isan aryl group having the attached radicals arranged in an ortho or paraconfiguration, which aryl group can be substituted or unsubstituted; R₃comprises the delivery-enhancing transporter; R₄ is substituted orunsubstituted S, O, N or C; R₅ is OH, SH or NHR₆; R₆ is hydrogen, alkyl,aryl, acyl or allyl; and k and m are each independently selected from 1and 2. In some embodiments, X is selected from the group consisting ofN, O, S, and CR₇R₈, wherein R₇ and R₈ are each independently selectedfrom the group consisting of H and alkyl. In some embodiments, R₄ is S;R₅ is NHR₆; and R₆ is hydrogen, methyl, allyl, butyl or phenyl. In someembodiments, the conjugate comprises:

[0024] In preferred embodiments, the compositions of the inventioncomprise a linker susceptible to solvent-mediated cleavage. For example,a preferred linker is substantially stable at acidic pH but issubstantially cleaved at physiological pH.

[0025] Additional embodiments of the invention provide transdermal drugformulations. These formulations include a therapeutically effectiveamount of a therapeutic agents a delivery-enhancing transporter thatincludes sufficient guanidino or amidino sidechain moieties to increasedelivery of the conjugate across one or more layers of an animalepithelial tissue compared to the trans-epithelial tissue delivery ofthe biologically active agent in non-conjugated form; and a vehiclesuited to transdermal drug administration.

BRIEF DESCRIPTION OF THE FIGURES

[0026]FIG. 1 shows a reaction scheme for the preparation of anα-chloroacetyl cyclosporin A derivative.

[0027]FIG. 2 shows a general procedure for the coupling, ofcysteine-containing peptides to the α-chloro acetyl cyclosporin Aderivative.

[0028]FIG. 3 shows a reaction scheme for the coupling of the cyclosporinA derivative to a biotin-labeled peptide.

[0029]FIG. 4 shows a reaction scheme for coupling of a cyclosporin Aderivative to an unlabeled peptide.

[0030] FIGS. 5A-H show various types of cleavable linkers that can beused to link a delivery-enhancing transporter to a biologically activeagent or other molecule of interest. FIG. 5A shows an example of adisulfide linkage. FIG. 5B shows a photocleavable linker which iscleaved upon exposure to electromagnetic radiation. FIG. 5C shows amodified lysyl residue used as a cleavable linker. FIG. 5D shows aconjugate in which the delivery-enhancing transporter T is linked to the2′-oxygen of the anticancer agent, paclitaxel. The linking moietyincludes (i) a nitrogen atom attached to the delivery-enhancingtransporter, (ii) a phosphate monoester located para to the nitrogenatom, and (iii) a carboxymethyl group meta to the nitrogen atom, whichis joined to the 2′-oxygen of paclitaxel by a carboxylate ester linkage.FIG. 5E a linkage of a delivery-enhancing transporter to a biologicallyactive agent, e.g., paclitaxel, by an aminoalkyl carboxylic acid; alinker amino group is joined to a delivery-enhancing transporter by anamide linkage and to a paclitaxel moiety by an ester linkage. FIGS. 5Fand G show chemical structures and conventional numbering of constituentbackbone atoms for paclitaxel and “TAXOTERE™” (R′=H, R″=BOC). FIG. 5Gshows the general chemical structure and ring atom numbering for taxoidcompounds.

[0031]FIG. 6 displays a synthetic scheme for a chemical conjugatebetween a heptamer of L-arginine and cyclosporin A (panel A) and its pHdependent chemical release (panel B). The α-chloro ester (2) was treatedwith benzylamine in the presence of sodium iodide to effectsubstitution, giving the secondary amine (5). Amine (5) was treated withanhydride (6) and the resultant crude acid (7) was converted to itscorresponding NHS ester (8). Ester (8) was then coupled with the aminoterminus of hepta-L-arginine, giving the N-Boc protected CsA conjugate(9). Finally, removal of the Boc protecting group with formic acidafforded the conjugate (10) as its octatrifluoroacetate salt after HPLCpurification.

[0032]FIG. 7 displays inhibition of inflammation in murine contactdermatitis by releasable R7 CsA. Balb/c (6-7 weeks) mice were painted onthe abdomen with 100 μl of 0.7% DNFB in acetone olive oil (95:5). Threedays later both ears of the animals were restimulated with 0.5% DNFB inacetone. Mice were treated one, five, and twenty hours afterrestimulation with either vehicle alone, 1% R7 peptide alone, 1% CsA, 1%nonreleasable R7 CsA, 0.01%/0.1%/1.0% releasable R7 CsA, and thefluorinated steroid positive control 0.1% triamcinolone acetonide. Earinflammation was measured 24 hours after restimulation using a springloaded caliper. The percent reduction of inflammation was calculatedusing the following formula (t−n)/(u−n), where t=thickness of thetreated ear, n=the thickness of a normal untreated ear, and u=thicknessof an inflamed ear without any treatment. N=20 animals in each group.

[0033]FIG. 8 shows a procedure for the preparation of acopper-diethylenetriaminepentaacetic acid complex (Cu-DTPA).

[0034]FIG. 9 shows a procedure for linking the Cu-DTPA to a transporterthrough an aminocaproic acid.

[0035]FIG. 10 shows a reaction for the acylation of hydrocortisone withchloroacetic anhydride.

[0036]FIG. 11 shows a reaction for linking the acylated hydrocortisoneto a transporter.

[0037]FIG. 12 shows a reaction for preparation of C-2′ derivatives oftaxol.

[0038]FIG. 13 shows a schematic of a reaction for coupling of a taxolderivative to a biotin-labeled peptide.

[0039]FIG. 14 shows a reaction for coupling of an unlabeled peptide to aC-2′ derivative of taxol.

[0040] FIGS. 15A-C shows a reaction scheme for the formation of otherC-2′ taxol-peptide conjugates.

[0041]FIG. 16 shows a general strategy for synthesis of a conjugate inwhich a drug or other biological agent is linked to a delivery-enhancingtransporter by a pH-releasable linker.

[0042]FIG. 17 shows a schematic diagram of a protocol for synthesizing ataxol 2′-chloroacetyl derivative.

[0043]FIG. 18 shows a strategy by which a taxol 2′-chloroacetylderivative is linked to an arginine heptamer delivery-enhancingtransporter.

[0044]FIG. 19 shows three additional taxol-r7 conjugates that can bemade using the reaction conditions illustrated in FIG. 18.

[0045]FIG. 20 shows the results of a 3 day MTT cytotoxicity assay usingtaxol and two different linkers.

[0046]FIG. 21: FACS cellular uptake assay of truncated analogs ofTat₄₉₋₅₇ (Fl-ahx-RKKRRQRRR): Tat₄₉₋₅₆ (Fl-ahx-RKKRRQRR), Tat₄₉₋₅₅(Fl-ahx-RKKRRQR), Tat₅₀₋₅₇ (Fl-ahx-KKRRQRRR), and Tat₅₁₋₅₇(Fl-ahx-KRRQRRR). Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of peptides for 15 min at 23° C.

[0047]FIG. 22 shows FACS cellular uptake assay of alanine-substitutedanalogs of Tat₄₉₋₅₇: A-49 (Fl-ahx-AKKRRQRRR), A-50 (Fl-ahx-RAKRRQRRR),A-51 (Fl-ahx-RKARRQRRR), A-52 (Fl-ahx-RKKARQRRR), A-53(Fl-ahx-RKKRAQRRR), A-54 (Fl-ahx-RKKRRARRR), A-55 (Fl-ahx-RKKRRQARR),A-56 (Fl-ahx-RKKRRQRAR), and A-57 (Fl-ahx-RKKRRQRRA). Jurkat cells wereincubated with varying concentrations (12.5 μM shown) of peptides for 12min at 23° C.

[0048]FIG. 23: FACS cellular uptake assay of d- and retro-isomers ofTat₄₉₋₅₇: d-Tat49-57 (Fl-ahx-rkkrrqrrr), Tat57-49 (Fl-ahx-RRRQRRKKR),and d-Tat57-49 (Fl-ahx-rrrqrrkkr). Jurkat cells were incubated withvarying concentrations (12.5 μM shown) of peptides for 15 min at 23° C.

[0049]FIG. 24: FACS cellular uptake of a series of arginine oligomersand Tat_(49≡): R5 (Fl-ahx-RRRRR), R6 (Fl-ahx-RRRRRR), R7(Fl-ahx-RRRRRRR), R8 (Fl-ahx-RRRRRRRR), R9 (Fl-ahx-RRRRRRRRR), r5(Fl-ahx-rrrrr), r6 (Fl-ahx-rrrrrr), r7 (Fl-ahx-rrrrrrr), r8(Fl-ahx-rrrrrrrr), r9 (Fl-ahx-rrrrrrrrr). Jurkat cells were incubatedwith varying concentrations (12.5 μM shown) of peptides for 4 min at 23°C.

[0050]FIG. 25: Preparation of guanidine-substituted peptoids.

[0051]FIG. 26: FACS cellular uptake of polyguanidine peptoids andd-arginine oligomers. Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of peptoids and peptides for 4 min at 23°C.

[0052]FIG. 27: FACS cellular uptake of d-arginine oligomers andpolyguanidine peptoids. Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of fluorescently labeled peptoids andpeptides for 4 min at 23° C.

[0053]FIG. 28: FACS cellular uptake of and d-arginine oligomers andN-hxg peptoids. Jurkat cells were incubated with varying concentrations(6.3 μM shown) of fluorescently labeled peptoids and peptides for 4 minat 23° C.

[0054]FIG. 29: FACS cellular uptake of d-arginine oligomers and N-chgpeptoids. Jurkat cells were incubated with varying concentrations (12.5μM shown) of fluorescently labeled peptoids and peptides for 4 min at23° C.

[0055]FIG. 30 shows a general strategy for attaching adelivery-enhancing transporter to a drug that includes a triazole ringstructure.

[0056]FIG. 31A and FIG. 31B show synthetic schemes for making conjugatesin which FK506 is attached to a delivery-enhancing transporter.

DETAILED DESCRIPTION

[0057] Definitions

[0058] An “epithelial tissue” is the basic tissue that covers surfaceareas of the surface, spaces, and cavities of the body. Epithelialtissues are composed primarily of epithelial cells that are attached toone another and rest on an extracellular matrix (basement membrane) thatis typically produced by the cells. Epithelial tissues include threegeneral types based on cell shape: squamous, cuboidal, and columnarepithelium. Squamous epithelium, which lines lungs and blood vessels, ismade up of flat cells. Cuboidal epithelium lines kidney tubules and iscomposed of cube shaped cells, while columnar epithelium cells line thedigestive tract and have a columnar appearance. Epithelial tissues canalso be classified based on the number of cell layers in the tissue. Forexample, a simple epithelial tissue is composed of a single layer ofcells, each of which sits on the basement membrane. A “stratified”epithelial tissue is composed of several cells stacked upon one another;not all cells contact the basement membrane. A “pseudostratified”epithelial tissue has cells that, although all contact the basementmembrane, appear to be stratified because the nuclei are at variouslevels.

[0059] The term “trans-epithelial” delivery or administration refers tothe delivery or administration of agents by permeation through one ormore layers of a body surface or tissue, such as intact skin or a mucousmembrane, by topical administration. Thus, the term is intended toinclude both transdermal (e.g., percutaneous adsorption) andtransmucosal administration. Delivery can be to a deeper layer of thetissue, for example, and/or delivery to the bloodstream.

[0060] “Delivery enhancement, “penetration enhancement” or “permeationenhancement” as used herein relates to an increase in amount and/or rateof delivery of a compound that is delivered into and across one or morelayers of an epithelial or endothelial tissue. An enhancement ofdelivery can be observed by measuring the rate and/or amount of thecompound that passes through one or more layers of animal or human skinor other tissue. Delivery enhancement also can involve an increase inthe depth into the tissue to which the compound is delivered, and/or theextent of delivery to one or more cell types of the epithelial or othertissue (e.g., increased delivery to fibroblasts, immune cells, andendothelial cells of the skin or other tissue). Such measurements arereadily obtained by, for example, using a diffusion cell apparatus asdescribed in U.S. Pat. No. 5,891,462.

[0061] The amount or rate of delivery of an agent across and/or intoskin or other epithelial or endothelial membrane is sometimesquantitated in terms of the amount of compound passing through apredetermined area of skin or other tissue, which is a defined area ofintact unbroken living skin or mucosal tissue. That area will usually bein the range of about 5 cm to about 100 cm, more usually in the range ofabout 10 cm to about 100 cm², still more usually in the range of about20 cm² to about 60 cm².

[0062] The terms “guanidyl,” guanidinyl” and “guanidino” are usedinterchangeably to refer to a moiety having the formula —HN═C(NH₂)NH(unprotonated form). As an example, arginine contains a guanidyl(guanidino) moiety, and is also referred to as2-amino-5-guanidinovaleric acid or α-amino-δ-guanidinovaleric acid.“Guanidium” refers to the positively charged conjugate acid form. Theterm “guanidino moiety” includes, for example, guanidine, guanidinium,guanidine derivatives such as (RNHC(NH)NHR′), monosubstitutedguanidines, monoguanides, biguanides, biguanide derivatives such as(RNHC(NH)NHC(NH)NHR′), and the like. In addition, the term “guanidinomoiety” encompasses any one or more of a guanide alone or a combinationof different guanides.

[0063] “Amidinyl” and “amidino” refer to a moiety having the formula—C(═NH)(NH₂). “Amidinium” refers to the positively charged conjugateacid form.

[0064] The term “trans-barrier concentration” or “trans-tissueconcentration” refers to the concentration of a compound present on theside of one or more layers of an epithelial or endothelial barriertissue that is opposite or “trans” to the side of the tissue to which aparticular composition has been added. For example, when a compound isapplied to the skin, the amount of the compound measured subsequentlyacross one or more layers of the skin is the trans-barrier concentrationof the compound.

[0065] “Biologically active agent” or “biologically active substance”refers to a chemical substance, such as a small molecule, macromolecule,or metal ion, that causes an observable change in the structure,function, or composition of a cell upon uptake by the cell. Observablechanges include increased or decreased expression of one or more mRNAs,increased or decreased expression of one or more proteins,phosphorylation of a protein or other cell component, inhibition oractivation of an enzyme, inhibition or activation of binding betweenmembers of a binding pair, an increased or decreased rate of synthesisof a metabolite, increased or decreased cell proliferation, and thelike.

[0066] The terms “therapeutic agent”, “therapeutic composition”, and“therapeutic substance” refer, without limitation, to any compositionthat can be used to the benefit of a mammalian species. Such agents maytake the form of ions, small organic molecules, peptides, proteins orpolypeptides, oligonucleotides, and oligosaccharides, for example.

[0067] The term “macromolecule” as used herein refers to large molecules(MW greater than 1000 daltons) exemplified by, but not limited to,peptides, proteins, oligonucleotides and polynucleotides of biologicalor synthetic origin.

[0068] “Small organic molecule” refers to a carbon-containing agenthaving a molecular weight (MW) of less than or equal to 1000 daltons.

[0069] The terms “non-polypeptide agent” and “non-polypeptidetherapeutic agent” refer to the portion of a conjugate that does notinclude the delivery-enhancing transporter, and that is a biologicallyactive agent other than a polypeptide. An example of a non-polypeptideagent is an anti-sense oligonucleotide, which can be conjugated to apolyarginine peptide to form a conjugate for enhanced delivery into andacross one or more layers of an epithelial or endothelial tissue.

[0070] A “subunit,” as used herein, is a monomeric unit that are joinedto form a larger polymeric compound. The set of amino acids are anexample of subunits. Each amino acid shares a common backbone (—C—C—N—),and the different amino acids differ in their sidechains. The backboneis repeated in a polypeptide. A subunit represents the shortestrepeating pattern of elements in a polymer backbone. For example, twoamino acids of a peptide are not considered a peptide because two aminoacids would not have the shortest repeating pattern of elements in thepolymer backbone.

[0071] The term “polymer” refers to a linear chain of two or moreidentical or non-identical subunits joined by covalent bonds. A peptideis an example of a polymer; peptides can be composed of identical ornon-identical amino acid subunits that are joined by peptide linkages(amide bonds).

[0072] The term “peptide” as used herein refers to a compound made up ofa single chain of D- or L-amino acids or a mixture of D- and L-aminoacids joined by peptide bonds. Generally, peptides contain at least twoamino acid residues and are less than about 50 amino acids in length.D-amino acids are represented herein by a lower-case one-letter aminoacid symbol (e.g., r for D-arginine), whereas L-amino acids arerepresented by an upper case one-letter amino acid symbol (e.g., R forL-arginine). Homopolymer peptides are represented by a one-letter aminoacid symbol followed by the number of consecutive occurrences of thatamino acid in the peptide—(e.g., R7 represents a heptamer that consistsof L-arginine residues).

[0073] The term “protein” as used herein refers to a corn-pound that iscomposed of linearly arranged amino acids linked by peptide bonds, butin contrast to peptides, has a well-defined conformation. Proteins, asopposed to peptides, generally consist of chains of 50 or more aminoacids.

[0074] “Polypeptide” as used herein refers to a polymer of at least twoamino acid residues and which contains one or more peptide bonds.“Polypeptide” encompasses peptides and proteins, regardless of whetherthe polypeptide has a well-defined conformation.

[0075] Description of the Preferred Embodiments

[0076] The present invention provides compositions and methods thatenhance the transfer of compounds, including drugs and otherbiologically active compounds, into and across one or more layers of ananimal epithelial or endothelial tissue. The methods involve contactingthe tissue with a conjugate that includes the compound of interestlinked to a delivery-enhancing transporter. The delivery enhancingtransporters provided by the invention are molecules that includesufficient guanidino or amidino moieties to increase delivery of theconjugate into and across one or more intact epithelial and endothelialtissue layers. The methods and compositions are useful fortrans-epithelial and trans-endothelial delivery of drugs and otherbiologically active molecules, and also for delivery of imaging anddiagnostic molecules. The methods and compositions of the invention areparticularly useful for delivery of compounds that requiretrans-epithelial or trans-endothelial transport to exhibit theirbiological effects, and that by themselves (without conjugation to adelivery-enhancing transporters or some other modification), are unable,or only poorly able, to cross such tissues and thus exhibit biologicalactivity.

[0077] The delivery-enhancing transporters and methods of the inventionprovide significant advantages over previously available methods forobtaining trans-epithelial and trans-endothelial tissue delivery ofcompounds of interest. The transporters make possible the delivery ofdrugs and other agents across tissues that were previously impenetrableto the drug. For example, while delivery of drugs across skin waspreviously nearly impossible for all but a few compounds, the methods ofthe invention can deliver compounds not only into cells of a first layerof an epithelial tissue such as skin, but also across one or more layersof the skin. The blood brain barrier is also resistant to transport ofdrugs and other diagnostic and therapeutic reagents; the methods andtransporters of the invention provide means to obtain such transport.

[0078] The delivery-enhancing transporters increase delivery of theconjugate into and across one or more intact epithelial or endothelialtissue layers compared to delivery of the compound in the absence of thedelivery-enhancing transporter. The delivery-enhancing transporters can,in some embodiments, increase delivery of the conjugate significantlyover that obtained using the tat protein of HIV-1 (Frankel et al. (1991)PCT Pub. No. WO 91/09958). Delivery is also increased significantly overthe use of shorter fragments of the tat protein containing the tat basicregion (residues 49-57 having the sequence RKKRRQRRR) (Barsoum et al.(1994) WO 94/04686 and Fawell et al. (1994) Proc. Nat'l. Acad. Sci. USA91: 664-668). Preferably, delivery obtained using the transporters ofthe invention is increased more than 2-fold, still more preferablysix-fold, still more preferably ten-fold, and still more preferablytwenty-fold, over that obtained with tat residues 49-57.

[0079] Similarly, the delivery-enhancing transporters of the inventioncan provide increased delivery compared to a 16 amino acidpeptide-cholesterol conjugate derived from the Antennapedia homeodomainthat is rapidly internalized by cultured neurons (Brugidou et al. (1995)Biochem. Biophys. Res. Commun. 214: 685-93). This region, residues 43-58at minimum, has the amino acid sequence RQIKIWFQNRRMKWKK. The Herpessimplex protein VP22, like tat and the Antennapedia domain, waspreviously known to enhance transport into cells, but was not known toenhance transport into and across endothelial and epithelial membranes(Elliot and O'Hare (1997) Cell 88: 223-33; Dilber et al. (1999) GeneTher. 6: 12-21; Phelan et al. (1998) Nature Biotechnol. 16: 440-3). Inpresently preferred embodiments, the delivery-enhancing transportersprovide significantly increased delivery compared to the Antennapediahomeodomain and to the VP22 protein.

[0080] Structure of Delivery-Enhancing Transporters

[0081] The delivery-enhancing transporters of the invention aremolecules that have sufficient guanidino and/or amidino moieties toincrease delivery of a compound to which the delivery-enhancingtransporter is attached into and across one or more layers of anepithelial tissue (e.g., skin or mucous membrane) or an endothelialtissue (e.g., the blood-brain barrier). The delivery-enhancingtransporters generally include a backbone structure to which is attachedthe guanidino and/or amidino sidechain moieties. In some embodiments,the backbone is a polymer that consists of subunits (e.g., repeatingmonomer units), at least some of which subunits contain a guanidino oramidino moiety.

[0082] A. Guanidino and/or Amidino Moieties

[0083] The delivery-enhancing transporters typically display at least 5guanidino and/or amidino moieties, and more preferably 7 or more suchmoieties. Preferably, the delivery-enhancing transporters have 25 orfewer guanidino and/or amidino moieties, and often have 15 or fewer ofsuch moieties. In some embodiments, the delivery-enhancing transporterconsists essentially of 50 or fewer subunits, and can consistessentially of 25 or fewer, 20 or fewer, or 15 or fewer subunits. Thedelivery-enhancing transporter can be as short as 5 subunits, in whichcase all subunits include a guanidino or amidino sidechain moiety. Thedelivery-enhancing transporters can have, for example, at least 6subunits, and in some embodiments have at least 7 or 10 subunits.Generally, at least 50% of the subunits contain a guanidino or amidinosidechain moiety. More preferably, at least 70% of the subunits, andsometimes at least 90% of the subunits in the delivery-enhancingtransporter contain a guanidino or amidino sidechain moiety.

[0084] Some or all of the guanidino and/or amidino moieties in thedelivery-enhancing transporters can be contiguous. For example, thedelivery-enhancing transporters can include from 6 to 25 contiguousguanidino and/or amidino-containing subunits. Seven or more contiguousguanidino and/or amidino-containing subunits are present in someembodiments. In some embodiments, each subunit that contains a guanidinomoiety is contiguous, as exemplified by a polymer containing at leastsix contiguous arginine residues.

[0085] The delivery-enhancing transporters are exemplified by peptides.Arginine residues or analogs of arginine can constitute the subunitsthat have a guanidino moiety. Such an arginine-containing peptide can becomposed of either all D-, all L- or mixed D- and L-amino acids, and caninclude additional amino acids, amino acid analogs, or other moleculesbetween the arginine residues. Optionally, the delivery-enhancingtransporter can also include a non-arginine residue to which a compoundto be delivered is attached, either directly or through a linker. Theuse of at least one D-arginine in the delivery-enhancing transporterscan enhance biological stability of the transporter during transit ofthe conjugate to its biological target. In some cases thedelivery-enhancing transporters are at least about 50% D-arginineresidues, and for even greater stability transporters in which all ofthe subunits are D-arginine residues are used. If the delivery enhancingtransporter molecule is a peptide, the transporter is not attached to anamino acid sequence to which the amino acids that make up the deliveryenhancing transporter molecule are attached in a naturally occurringprotein.

[0086] Preferably, the delivery-enhancing transporter is linear. In apreferred embodiment, an agent to be delivered into and across one ormore layers of an epithelial tissue is attached to a terminal end of thedelivery-enhancing transporter. In some embodiments, the agent is linkedto a single transport polymer to form a conjugate. In other embodiments,the conjugate can include more than one delivery-enhancing transporterlinked to an agent, or multiple agents linked to a singledelivery-enhancing transporter.

[0087] More generally, it is preferred that each subunit contains ahighly basic sidechain moiety which (i) has a pKa of greater than 11,more preferably 12.5 or greater, and (ii) contains, in its protonatedstate, at least two geminal amino groups (NH₂) which share aresonance-stabilized positive charge, which gives the moiety a bidentatecharacter.

[0088] The guanidino or amidino moieties extend away from the backboneby virtue of being linked to the backbone by a sidechain linker. Thesidechain atoms are preferably provided as methylene carbon atoms,although one or more other atoms such as oxygen, sulfur or nitrogen canalso be present. For example, a linker that attaches a guanidino moietyto a backbone can be shown as:

[0089] In these formulae, n is preferably at least 2, and is preferablybetween 2 and 7. In some embodiments, n is 3 (arginine for structure 1).In other embodiments, n is between 4 and 6; most preferably n is 5 or 6.Although the sidechain in the exemplified formulae is shown as beingattached to a peptide backbone (i.e., a repeating amide to which thesidechain is attached to the carbon atom that is α to the carbonylgroup, subunit 1) and a peptoid backbone (i.e., a repeating amide towhich the sidechain is attached to the nitrogen atom that is β to thecarbonyl group, subunit 2), other non-peptide backbones are alsosuitable, as discussed in more detail herein. Thus, similar sidechainlinkers can be attached to nonpeptide backbones (e.g., peptoidbackbones).

[0090] In some embodiments, the delivery-enhancing transporters arecomposed of linked subunits, at least some of which include a guanidinoand/or amidino moiety. Examples of suitable subunits having guanidinoand/or amidino moieties are described below.

[0091] Amino acids. In some embodiments, the delivery-enhancingtransporters are composed of D or L amino acid residues. The amino acidscan be naturally occurring or non-naturally occurring amino acids.Arginine (α-amino-δ-guanidinovaleric acid) andα-amino-ε-amidino-hexanoic acid (isosteric amidino analog) are examplesof suitable guanidino- and amidino-containing amino acid subunits. Theguanidinium group in arginine has a pKa of about 12.5. In some preferredembodiments the transporters are comprised of at least six contiguousarginine residues.

[0092] Other amino acids, such as α-amino-β-guanidino-propionic acid,α-amino-γ-guanidino-butyric acid, or α-amino-ε-guanidino-caproic acid(containing 2, 3 or 5 sidechain linker atoms, respectively, between thebackbone chain and the central guanidinium carbon) can also be used.

[0093] D-amino acids can also be used in the delivery enhancingtransporters. Compositions containing exclusively D-amino acids have theadvantage of decreased enzymatic degradation. However, they can alsoremain largely intact within the target cell. Such stability isgenerally not problematic if the agent is biologically active when thepolymer is still attached. For agents that are inactive in conjugateform, a linker that is cleavable at the site of action (e.g., by enzyme-or solvent-mediated cleavage within a cell) should be included withinthe conjugate to promote release of the agent in cells or organelles.

[0094] Other Subunits. Subunits other than amino acids can also beselected for use in forming transport polymers. Such subunits caninclude, but are not limited to, hydroxy amino acids, N-methyl-aminoacids amino aldehydes, and the like, which result in polymers withreduced peptide bonds. Other subunit types can be used, depending on thenature of the selected backbone, as discussed in the next section.

[0095] B. Backbones

[0096] The guanidino and/or amidino moieties that are included in thedelivery-enhancing transporters are generally attached to a linearbackbone. The backbone can comprise a variety of atom types, includingcarbon, nitrogen, oxygen, sulfur and phosphorus, with the majority ofthe backbone chain atoms typically consisting of carbon. A plurality ofsidechain moieties that include a terminal guanidino or amidino groupare attached to the backbone. Although spacing between adjacentsidechain moieties is typically consistent, the delivery-enhancingtransporters used in the invention can also include variable spacingbetween sidechain moieties along the backbone.

[0097] A more detailed backbone list includes N-substituted amide (CONRreplaces CONH linkages), esters (CO₂), keto-methylene (COCH₂) reduced ormethyleneamino (CH₂NH), thioamide (CSNH), phosphinate (PO₂RCH₂),phosphonamidate and phosphonamidate ester (PO₂RNH), retropeptide (NHCO),trans-alkene (CR═CH), fluoroalkene (CF═CH), dimethylene (CH₂CH₂),thioether (CH₂S), hydroxyethylene (CH(OH)CH₂), methyleneoxy (CH₂O),tetrazole (CN₄), retrothioamide (NHCS), retroreduced (NHCH₂),sulfonamido (SO₂NH), methylenesulfonamido (CHRSO₂NH), retrosulfonamide(NHSO₂), and peptoids (N-substituted amides), and backbones withmalonate and/or gem-diamino-alkyl subunits, for example, as reviewed byFletcher et al. ((1998) Chem. Rev. 98:763) and detailed by referencescited therein. Many of the foregoing substitutions result inapproximately isosteric polymer backbones relative to backbones formedfrom α-amino acids.

[0098] As mentioned above, in a peptoid backbone, the sidechain isattached to the backbone nitrogen atoms rather than the carbon atoms.(See e.g., Kessler (1993) Angew. Chem. Int. Ed. Engl. 32:543; Zuckermanet al. (1992) Chemtracts-Macromol. Chem. 4:80; and Simon et al. (1992)Proc. Nat'l. Acad. Sci. USA 89:9367.) An example of a suitable peptoidbackbone is poly-(N-substituted)glycine (poly-NSG). Synthesis ofpeptoids is described in, for example, U.S. Pat. No. 5,877,278. As theterm is used herein, transporters that have a peptoid backbone areconsidered “non-peptide” transporters, because the transporters are notcomposed of amino acids having naturally occurring sidechain locations.Non-peptoid backbones, including peptoid backbones, provide enhancedbiological stability (for example, resistance to enzymatic degradationin vivo).

[0099] C. Synthesis of Delivery-enhancing Transporters

[0100] Delivery-enhancing transporters are constructed by any methodknown in the art. Exemplary peptide polymers can be producedsynthetically, preferably using a peptide synthesizer (e.g., an AppliedBiosystems Model 433) or can be synthesized recombinantly by methodswell known in the art. Recombinant synthesis is generally used when thedelivery enhancing transporter is a peptide which is fused to apolypeptide or protein of interest.

[0101] N-methyl and hydroxy-amino acids can be substituted forconventional amino acids in solid phase peptide synthesis. However,production of delivery-enhancing transporters with reduced peptide bondsrequires synthesis of the dimer of amino acids containing the reducedpeptide bond. Such dimers are incorporated into polymers using standardsolid phase synthesis procedures. Other synthesis procedures are wellknown and can be found, for example, in Fletcher et al. (1998) Chem.Rev. 98:763, Simon et al. (1992) Proc. Nat'l. Acad. Sci. USA 89:9367,and references cited therein.

[0102] The delivery-enhancing transporters of the invention can beflanked by one or more non-guanidino/non-amidino subunits (such asglycine, alanine, and cysteine, for example), or a linker (such as anaminocaproic acid group), that do not significantly affect the rate oftrans-tissue layer transport of the corresponding delivery-enhancingtransporter-containing conjugates. Also, any free amino terminal groupcan be capped with a blocking group, such as an acetyl or benzyl group,to prevent ubiquitination in vivo.

[0103] Where the transporter is a peptoid polymer, one synthetic methodinvolves the following steps: 1) a peptoid polyamine is treated with abase and pyrazole-1-carboxamidine to provide a mixture; 2) the mixtureis heated and then allowed to cool; 3) the cooled mixture is acidified;and 4) the acidified mixture is purified. Preferably the base used instep 1 is a carbonate, such as sodium carbonate, and heating step 2involves heating the mixture to approximately 50° C. for between about24 hours and about 48 hours. The purification step preferably involveschromatography (e.g., reverse-phase HPLC).

[0104] D. Attachment of Transport Polymers To Biologically Active Agents

[0105] The agent to be transported can be linked to thedelivery-enhancing transporter according to a number of embodiments. Inone embodiment, the agent is linked to a single delivery-enhancingtransporter, either via linkage to a terminal end of thedelivery-enhancing transporter or to an internal subunit within thereagent via a suitable linking group.

[0106] In a second embodiment, the agent is attached to more than onedelivery-enhancing transporter, in the same manner as above. Thisembodiment is somewhat less preferred, since it can lead to crosslinkingof adjacent cells.

[0107] In a third embodiment, the conjugate contains two agent moietiesattached to each terminal end of the delivery-enhancing transporter. Forthis embodiment, it is presently preferred that the agent has amolecular weight of less than 10 kDa.

[0108] With regard to the first and third embodiments just mentioned,the agent is generally not attached to one any of the guanidino oramidino sidechains so that they are free to interact with the targetmembrane.

[0109] The conjugates of the invention can be prepared bystraightforward synthetic schemes. Furthermore, the conjugate productsare usually substantially homogeneous in length and composition, so thatthey provide greater consistency and reproducibility in their effectsthan heterogeneous mixtures.

[0110] According to an important aspect of the present invention, it hasbeen found by the applicants that attachment of a singledelivery-enhancing transporter to any of a variety of types ofbiologically active agents is sufficient to substantially enhance therate of uptake of an agent into and across one or more layers ofepithelial and endothelial tissues, even without requiring the presenceof a large hydrophobic moiety in the conjugate. In fact, attaching alarge hydrophobic moiety can significantly impede or prevent cross-layertransport due to adhesion of the hydrophobic moiety to the lipid bilayerof cells that make up the epithelial or endothelial tissue. Accordingly,the present invention includes conjugates that do not containsubstantially hydrophobic moieties, such as lipid and fatty acidmolecules.

[0111] Delivery-enhancing transporters of the invention can be attachedcovalently to biologically active agents by chemical or recombinantmethods.

[0112] 1. Chemical Linkages

[0113] Biologically active agents such as small organic molecules andmacromolecules can be linked to delivery-enhancing transporters of theinvention via a number of methods known in the art (see, for example,Wong, S. S., Ed., Chemistry of Protein Conjugation and Cross-Linking,CRC Press, Inc., Boca Raton, Fla. (1991), either directly (e.g., with acarbodiimide) or via a linking moiety. In particular, carbamate, ester,thioether, disulfide, and hydrazone linkages are generally easy to formand suitable for most applications. Ester and disulfide linkages arepreferred if the linkage is to be readily degraded in the cytosol, aftertransport of the substance across the cell membrane.

[0114] Various functional groups (hydroxyl, amino, halogen, etc.) can beused to attach the biologically active agent to the transport polymer.Groups which are not known to be part of an active site of thebiologically active agent are preferred, particularly if the polypeptideor any portion thereof is to remain attached to the substance afterdelivery.

[0115] Polymers, such as peptides produced as described in PCTapplication US98/10571 (Publication No. WO 9852614), are generallyproduced with an amino terminal protecting group, such as FMOC. Forbiologically active agents which can survive the conditions used tocleave the polypeptide from the synthesis resin and deprotect thesidechains, the FMOC may be cleaved from the N-terminus of the completedresin-bound polypeptide so that the agent can be linked to the freeN-terminal amine. In such cases, the agent to be attached is typicallyactivated by methods well known in the art to produce an active ester oractive carbonate moiety effective to form an amide or carbamate linkage,respectively, with the polymer amino group. Of course, other linkingchemistries can also be used.

[0116] To help minimize side-reactions, guanidino and amidino moietiescan be blocked using conventional protecting groups, such ascarbobenzyloxy groups (CBZ), di-t-BOC, PMC, Pbf, N-NO₂, and the like.

[0117] Coupling reactions are performed by known coupling methods in anyof an array of solvents, such as N,N-dimethyl formamide (DMF), N-methylpyrrolidinone, dichloromethane, water, and the like. Exemplary couplingreagents include, for example, O-benzotriazolyloxy tetramethyluroniumhexafluorophosphate (HATU), dicyclohexyl carbodiimide,bromo-tris(pyrrolidino) phosphonium bromide (PyBroP), etc. Otherreagents can be included, such as N,N-dimethylamino pyridine (DMAP),4-pyrrolidino pyridine, N-hydroxy succinimide, N-hydroxy benzotriazole,and the like.

[0118] 2. Fusion Polypeptides

[0119] Delivery-enhancing transporters of the invention can be attachedto biologically active polypeptide agents by recombinant means byconstructing vectors for fusion proteins comprising the polypeptide ofinterest and the delivery-enhancing transporter, according to methodswell known in the art. Generally, the delivery-enhancing transportercomponent will be attached at the C-terminus or N-terminus of thepolypeptide of interest, optionally via a short peptide linker.

[0120] 3. Releasable Linkers

[0121] The biologically active agents are, in presently preferredembodiments, attached to the delivery-enhancing transporter using alinkage that is specifically cleavable or releasable. The use of suchlinkages is particularly important for biologically active agents thatare inactive until the attached delivery-enhancing transporter isreleased. In some cases, such conjugates that consist of a drug moleculethat is attached to a delivery-enhancing transporter can be referred toas prodrugs, in that the release of the delivery-enhancing transporterfrom the drug results in conversion of the drug from an inactive to anactive form. As used herein, “cleaved” or “cleavage” of a conjugate orlinker refers to release of a biological agent from a transportermolecule, thereby releasing an active biological agent. “Specificallycleavable” or “specifically releasable” refers to the linkage betweenthe transporter and the agent being cleaved, rather than the transporterbeing degraded (e.g., by proteolytic degradation).

[0122] In some embodiments, the linkage is a readily cleavable linkage,meaning that it is susceptible to cleavage under conditions found invivo. Thus, upon passing into and through one or more layers of anepithelial and/or endothelial tissue, the agent is released from thedelivery-enhancing transporter. Readily cleavable linkages can be, forexample, linkages that are cleaved by an enzyme having a specificactivity (e.g., an esterase, protease, phosphatase, peptidase, and thelike) or by hydrolysis. For this purpose, linkers containing carboxylicacid esters and disulfide bonds are sometimes preferred, where theformer groups are hydrolyzed enzymatically or chemically, and the latterare severed by disulfide exchange, e.g., in the presence of glutathione.The linkage can be selected so it is cleavable by an enzymatic activitythat is known to be present in one or more layers of an epithelial orendothelial tissue. For example, the stratum granulosum of skin has arelatively high concentration of N-peptidase activity.

[0123] A specifically cleavable linker can be engineered onto atransporter molecule. For example, amino acids that constitute aprotease recognition site, or other such specifically recognizedenzymatic cleavage site, can be used to link the transporter to theagent. Altematively, chemical or other types of linkers that arecleavable by, for example, exposure to light or other stimulus can beused to link the transporter to the agent of interest.

[0124] A conjugate in which an agent to be delivered and adelivery-enhancing transporter are linked by a specifically cleavable orspecifically releasable linker will have a half-life. The term“half-life” in this context refers to the amount of time required afterapplying the conjugate to an epithelial or endothelial membrane for onehalf of the amount of conjugate to become dissociated to release thefree agent. The half-life for some embodiments is typically between 5minutes and 24 hours, and more preferably is between 30 minutes and 2hours. The half-life of a conjugate can be “tuned” or modified,according to the invention, as described below.

[0125] In some embodiments, the cleavage rate of the linkers is pHdependent. For example, a linker can form a stable linkage between anagent and a delivery-enhancing transporter at an acidic pH (e.g., pH 6.5or less, more preferably about 6 or less, and still more preferablyabout 5.5 or less). However, when the conjugate is placed atphysiological pH (e.g., pH 7 or greater, preferably about pH 7.4), thelinker will undergo cleavage to release the agent. Such pH sensitivitycan be obtained by, for example, including a functional group that, whenprotonated (i.e., at an acidic pH), does not act as a nucleophile. At ahigher (e.g., physiological) pH, the functional group is no longerprotonated and thus can act as a nucleophile. Examples of suitablefunctional groups include, for example, N and S. One can use suchfunctional groups to fine-tune the pH at which self-cleavage occurs.

[0126] The cleavable linker can be self-immolating. Such linkers containa nucleophile (e.g., oxygen, nitrogen or sulfur) distal to the agent anda cleavable group (e.g., ester, carbonate, carbamate, thiocarbamate)proximal to the agent. Intramolecular attack of the nucleophile on thecleavable group either directly or indirectly releases the agent. Ingeneral, the nucleophile is 5 to 6 atoms distal from the cleaved group,thereby forming a 5-6 member ring as a product of immolation.

[0127] Such linkers include those having a structure 3, 4, or 5, asfollows:

[0128] wherein:

[0129] R₁—X comprises the agent to be delivered;

[0130] X is a functional group on the agent, to which functional groupthe linker is attached;

[0131] Y is N or C;

[0132] R₂ is hydrogen, alkyl, aryl, acyl, or allyl;

[0133] R₃ comprises the delivery-enhancing transporter;

[0134] R₄ is substituted or unsubstituted S, O, N or C;

[0135] R₅ is OH, SH or NHR₆;

[0136] R₆ is hydrogen, alkyl, aryl, acyl or allyl;

[0137] k and m are each independently selected from 1 and 2; and

[0138] n is 1 to 10.

[0139] The agent to be delivered (e.g., a drug or diagnostic agent)generally includes a functional group (designated as X in the formulaeabove) by which the linker is attached to the delivery-enhancingtransporter. Examples of suitable functional groups for X include, forexample, N, O, S, and CR₇R₈, wherein R₇ and R₈ are each independentlyselected from the group consisting of H and alkyl. If X is O, forexample, release of the agent from the delivery-enhancing transporterand linker will yield the agent in its free alcohol form; if X is N, thefree amine will result. Similarly, if X is S, release of the linker willyield the agent in the thiol form.

[0140] For a linker having the structure 3, one can adjust the half-lifeof a conjugate that includes the linker by the choice of the R₂substituent. By using an R₂ of increased or decreased size, one canobtain a conjugate that has a longer or shorter half-life, respectively.R₂ is preferably methyl, ethyl, propyl, butyl, allyl, benzyl or phenyl.Similar modulation of half-life can be accomplished in an analogousmanner with other linkers (e.g., R₅ on structure 4).

[0141] Structure 3 also provides an example of how the linker structurecan affect the pH sensitivity of a conjugate. The backbone amino groupis protonated at acidic pH (e.g., pH 5.5), and thus is stable in thatthe amine does not function as a strong nucleophile. Upon raising the pHto physiological pH (e.g., pH 7.4), however, the amine is no longerprotonated and thus can act as a nucleophile. The resulting nucleophilicattack by the amine upon the carbonyl adjacent to the agent of interestthen results in release of the agent from the linker anddelivery-enhancing transporter. Again, this rationale also applies toother linker structures. In an example of a preferred linker ofstructure 3, R₂ is benzyl; k, m, and n are each 1, and X is O.

[0142] An example of a suitable linker of structure 4 has R₅ as NHR₆; R₆selected from hydrogen, methyl, allyl, butyl or phenyl; and k and m eachbeing 1.

[0143] Another example of a self-immolating linker is represented bystructure 5 as shown above. In presently preferred conjugates thatinclude linkers of this structure, R₄ is S and R₅ is NHR₆, wherein R ishydrogen, methyl, allyl, butyl or phenyl; and k is 2.

[0144] For example, a presently preferred conjugate comprises thestructure:

[0145] wherein Ph is phenyl.

[0146] The invention also provides linkers in which cleavage occurs intwo stages. The first stage of cleavage is rate-limiting and can also befine-tuned for pH sensitivity and half-life. Once this initialrearrangement has occurred, the second stage of the intramolecularreaction occurs relatively quickly. An example of a conjugate havingthis type of linker is represented as structure 6:

[0147] wherein:

[0148] R₁—X is the agent to be delivered;

[0149] X is a functional group on the agent, to which functional groupthe linker is attached;

[0150] Ar is an aryl group having the attached radicals arranged in anortho or para configuration, which aryl group can be substituted orunsubstituted;

[0151] R₃ is the delivery-enhancing transporter;

[0152] R₄ is substituted or unsubstituted S, O, N or C;

[0153] R₅ is OH, SH or NHR₆;

[0154] R₆ is hydrogen, alkyl, aryl, acyl or allyl; and

[0155] k and m are each independently selected from 1 and 2.

[0156] Examples of preferred conjugates of structure 6 include those inwhich R₄ is S, R₅ is NHR₆, and R₆ is hydrogen, methyl, allyl, butyl orphenyl. For example, a suitable conjugate of structure 6 is:

[0157] The self-immolating linkers typically undergo intramolecularcleavage with a half-life between about 10 minutes and about 24 hours inwater at a pH of approximately 7.4. Preferably, the cleavage half-lifeis between about 20 minutes and about 4 hours in water at a pH ofapproximately 7.4. More preferably, the cleavage half-life is betweenabout 30 minutes and about 2 hours in water at a pH of approximately7.4.

[0158] In another preferred embodiment, the cleavable linker contains afirst cleavable group that is distal to the agent, and a secondcleavable group that is proximal to the agent, such that cleavage of thefirst cleavable group yields a linker-agent conjugate containing anucleophilic moiety capable of reacting intramolecularly to cleave thesecond cleavable group, thereby releasing the agent from the linker andpolymer. This embodiment is illustrated by various small moleculeconjugates discussed below and in PCT application US98/10571(Publication No. WO 9852614).

[0159] In one approach, the conjugate can include a disulfide linkage,as illustrated in FIG. 5A (see also, PCT application US98/10571(Publication No. WO 9852614)), which shows a conjugate (I) containing atransport polymer T which is linked to a cytotoxic agent,6-mercaptopurine, by an N-acetyl-protected cysteine group which servesas a linker. Thus, the cytotoxic agent is attached by a disulfide bondto the 6-mercapto group, and the transport polymer is bound to thecysteine carbonyl moiety via an amide linkage. Cleavage of the disulfidebond by reduction or disulfide exchange results in release of the freecytotoxic agent.

[0160] A method for synthesizing a disulfide-containing conjugate isprovided in Example 9A of PCT application US98/10571. The productcontains a heptamer of Arg residues which is linked to 6-mercaptopurineby an N-acetyl-Cys-Ala-Ala linker, where the Ala residues are includedas an additional spacer to render the disulfide more accessible tothiols and reducing agents for cleavage within a cell. The linker inthis example also illustrates the use of amide bonds, which can becleaved enzymatically within a cell.

[0161] In another approach, the conjugate includes a photocleavablelinker which is cleaved upon exposure to electromagnetic radiation. Anexemplary linkage is illustrated in FIG. 5B, which shows a conjugate(II) containing a transport polymer T which is linked to6-mercaptopurine via a meta-nitrobenzoate linking moiety. Polymer T islinked to the nitrobenzoate moiety by an amide linkage to the benzoatecarbonyl group, and the cytotoxic agent is bound via its 6-mercaptogroup to the p-methylene group. The compound can be formed by reacting6-mercaptopurine with p-bromomethyl-m-nitrobenzoic acid in the presenceof NaOCH₃/methanol with heating, followed by coupling of the benzoatecarboxylic acid to a transport polymer, such as the amino group of aγ-aminobutyric acid linker attached to the polymer (see e.g., Example 9Bof PCT application US98/10571). Photo-illumination of the conjugatecauses release of the 6-mercaptopurine by virtue of the nitro group thatis ortho to the mercaptomethyl moiety. This approach finds utility inphototherapy methods as are known in the art, particularly forlocalizing drug activation to a selected area of the body.

[0162] Preferably, the cleavable linker contains first and secondcleavable groups that can cooperate to cleave the polymer from thebiologically active agent, as illustrated by the following approaches.That is, the cleavable linker contains a first cleavable group that isdistal to the agent, and a second cleavable group that is proximal tothe agent, such that cleavage of the first cleavable group yields alinker-agent conjugate containing a nucleophilic moiety capable ofreacting intramolecularly to cleave the second cleavable group, therebyreleasing the agent from the linker and polymer:

[0163]FIG. 5C shows a conjugate (III) containing a transport polymer Tlinked to the anticancer agent, 5-fluorouracil (5FU). Here, the linkageis provided by a modified lysyl residue. The transport polymer is linkedto the α-amino group, and the 5-fluorouracil is linked via theα-carbonyl. The lysyl ε-amino group has been modified to a carbamateester of o-hydroxymethyl nitrobenzene, which comprises a first,photolabile cleavable group in the conjugate. Photo-illumination seversthe nitrobenzene moiety from the conjugate, leaving a carbamate thatalso rapidly decomposes to give the free α-amino group, an effectivenucleophile. Intramolecular reaction of the ε-amino group with the amidelinkage to the 5-fluorouracil group leads to cyclization with release ofthe 5-fluorouracil group.

[0164]FIG. 5D illustrates a conjugate (IV) containing adelivery-enhancing transporter T linked to 2′-oxygen of the anticanceragent, paclitaxel. The linkage is provided by a linking moiety thatincludes (i) a nitrogen atom attached to the delivery-enhancingtransporter, (ii) a phosphate monoester located para to the nitrogenatom, and (iii) a carboxymethyl group meta to the nitrogen atom, whichis joined to the 2′-oxygen of paclitaxel by a carboxylate ester linkage.Enzymatic cleavage of the phosphate group from the conjugate affords afree phenol hydroxyl group. This nucleophilic group then reactsintramolecularly with the carboxylate ester to release free paclitaxel,fully capable of binding to its biological target. Example 9C of PCTapplication US98/10571 describes a synthetic protocol for preparing thistype of conjugate.

[0165]FIG. 5E illustrates yet another approach wherein adelivery-enhancing transporter is linked to a biologically active agent,e.g., paclitaxel, by an aminoalkyl carboxylic acid. Preferably, thelinker amino group is linked to the linker carboxyl carbon by from 3 to5 chain atoms (n=3 to 5), preferably either 3 or 4 chain atoms, whichare preferably provided as methylene carbons. As seen in FIG. 5E, thelinker amino group is joined to the delivery-enhancing transporter by anamide linkage, and is joined to the paclitaxel moiety by an esterlinkage. Enzymatic cleavage of the amide linkage releases thedelivery-enhancing transporter and produces a free nucleophilic aminogroup. The free amino group can then react intramolecularly with theester group to release the linker from the paclitaxel.

[0166] In another approach, the conjugate includes a linker that islabile at one pH but is stable at another pH. For example, FIG. 6illustrates a method of synthesizing a conjugate with a linker that iscleaved at physiological pH but is stable at acidic pH. Preferably, thelinker is cleaved in water at a pH of from about 6.6 to about 7.6.Preferably the linker is stable in water at a pH from about 4.5 to about6.5.

[0167] Uses of Delivery-enhancing Transporters

[0168] The delivery-enhancing transporters find use in therapeutic,prophylactic and diagnostic applications. The delivery-enhancingtransporters can carry a diagnostic or biologically active reagent intoand across one or more layers of skin or other epithelial tissue (e.g.,gastrointestinal, lung, and the like), or across endothelial tissuessuch as the blood brain barrier. This property makes the reagents usefulfor treating conditions by delivering agents that must penetrate acrossone or more tissue layers in order to exert their biological effect.

[0169] Compositions and methods of the present invention have particularutility in the area of human and veterinary therapeutics. Generally,administered dosages will be effective to deliver picomolar tomicromolar concentrations of the therapeutic composition to the effectorsite. Appropriate dosages and concentrations will depend on factors suchas the therapeutic composition or drug, the site of intended delivery,and the route of administration, all of which can be derived empiricallyaccording to methods well known in the art. Further guidance can beobtained from studies using experimental animal models for evaluatingdosage, as are known in the art.

[0170] Administration of the compounds of the invention with a suitablepharmaceutical excipient as necessary can be carried out via any of theaccepted modes of administration. Thus, administration can be, forexample, intravenous, topical, subcutaneous, transcutaneous,intramuscular, oral, intra-joint, parenteral, peritoneal, intranasal, orby inhalation. Suitable sites of administration thus include, but arenot limited to, skin, bronchial, gastrointestinal, anal, vaginal, eye,and ear. The formulations may take the form of solid, semi-solid,lyophilized powder, or liquid dosage forms, such as, for example,tablets, pills, capsules, powders, solutions, suspensions, emulsions,suppositories, retention enemas, creams, ointments, lotions, aerosols orthe like, preferably in unit dosage forms suitable for simpleadministration of precise dosages.

[0171] The compositions typically include a conventional pharmaceuticalcarrier or excipient and may additionally include other medicinalagents, carriers, adjuvants, and the like. Preferably, the compositionwill be about 5% to 75% by weight of a compound or compounds of theinvention, with the remainder consisting of suitable pharmaceuticalexcipients. Appropriate excipients can be tailored to the particularcomposition and route of administration by methods well known in theart, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., MackPublishing Co., Easton, Pa. (1990).

[0172] For oral administration, such excipients include pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesiumcarbonate, and the like. The composition may take the form of asolution, suspension, tablet, pill, capsule, powder, sustained-releaseformulation, and the like.

[0173] In some embodiments, the pharmaceutical compositions take theform of a pill, tablet or capsule, and thus, the composition cancontain, along with the biologically active conjugate, any of thefollowing: a diluent such as lactose, sucrose, dicalcium phosphate, andthe like; a disintegrant such as starch or derivatives thereof; alubricant such as magnesium stearate and the like; and a binder such astarch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose andderivatives thereof.

[0174] The active compounds of the formulas may be formulated into asuppository comprising, for example, about 0.5% to about 50% of acompound of the invention, disposed in a polyethylene glycol (PEG)carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]).

[0175] Liquid compositions can be prepared by dissolving or dispersingcompound (about 0.5% to about 20%), and optional pharmaceuticaladjuvants in a carrier, such as, for example, aqueous saline (e.g., 0.9%w/v sodium chloride), aqueous dextrose, glycerol, ethanol and the like,to form a solution or suspension, e.g., for intravenous administration.The active compounds may also be formulated into a retention enema.

[0176] If desired, the composition to be administered may also containminor amounts of non-toxic auxiliary substances such as wetting oremulsifying agents, pH buffering agents, such as, for example, sodiumacetate, sorbitan monolaurate, or triethanolamine oleate.

[0177] For topical administration, the composition is administered inany suitable format, such as a lotion or a transdermal patch. Fordelivery by inhalation, the composition can be delivered as a dry powder(e.g., Inhale Therapeutics) or in liquid form via a nebulizer.

[0178] Methods for preparing such dosage forms are known or will beapparent to those skilled in the art; for example, see Remington'sPharmaceutical Sciences, supra., and similar publications. Thecomposition to be administered will, in any event, contain a quantity ofthe pro-drug and/or active compound(s) in a pharmaceutically effectiveamount for relief of the condition being treated when administered inaccordance with the teachings of this invention.

[0179] Generally, the compounds of the invention are administered in atherapeutically effective amount, i.e., a dosage sufficient to effecttreatment, which will vary depending on the individual and conditionbeing treated. Typically, a therapeutically effective daily dose is from0.1 to 100 mg/kg of body weight per day of drug. Most conditions respondto administration of a total dosage of between about 1 and about 30mg/kg of body weight per day, or between about 70 mg and 2100 mg per dayfor a 70 kg person.

[0180] Stability of the conjugate can be further controlled by thecomposition and stereochemistry of the backbone and sidechains of thedelivery-enhancing transporters. For polypeptide delivery-enhancingtransporters, D-isomers are generally resistant to endogenous proteases,and therefore have longer half-lives in serum and within cells.D-polypeptide polymers are therefore appropriate when longer duration ofaction is desired. L-polypeptide polymers have shorter half-lives due totheir susceptibility to proteases, and are therefore chosen to impartshorter acting effects. This allows side-effects to be averted morereadily by withdrawing therapy as soon as side-effects are observed.Polypeptides comprising mixtures of D and L-residues have intermediatestabilities. Homo-D-polymers are generally preferred.

[0181] A. Application to Skin

[0182] The delivery-enhancing transporters of the invention makepossible the delivery of biologically active and diagnostic agentsacross the skin. Surprisingly, the transporters can deliver an agentacross the stratum corneum, which previously had been a nearlyimpenetrable barrier to drug delivery. The stratum corneum, theoutermost layer of the skin, is composed of several layers of dead,keratin-filled skin cells that are tightly bound together by a “glue”composed of cholesterol and fatty acids. Once the agents are deliveredthrough the stratum corneum by the transporters of the invention, theagents can enter the viable epidermis, which is composed of the stratumgranulosum, stratum lucidum and stratum germinativum which, along withthe stratum corneum, make up the epidermis. Delivery in some embodimentsof the invention is through the epidermis and into the dermis, includingone or both of the papillary dermis and the reticular dermis.

[0183] This ability to obtain penetration of one or more layers of theskin can greatly enhance the efficacy of compounds such asantibacterials, antifungals, antivirals, antiproliferatives,immunosuppressives, vitamins, analgesics, hormones, and the like.Numerous such compounds are known to those of skill in the art (see,e.g., Hardman and Limbird, Goodman & Gilman's The Pharmacological Basisof Therapeutics, McGraw-Hill, New York, 1996).

[0184] In some embodiments, the agent is delivered into a blood vesselthat is present in the epithelial tissue, thus providing a means fordelivery of the agent systemically. Delivery can be eitherintrafollicular or interfollicular, or both. Pretreatment of the skin isnot required for delivery of the conjugates.

[0185] In other embodiments, the delivery-enhancing transporters areuseful for delivering cosmetics and agents that can treat skinconditions. Target cells in the skin that are of interest include, forexample, fibroblasts, epithelial cells and immune cells. For example,the transporters provide the ability to deliver compounds such asantiinflammatory agents to immune cells found in the dermis.

[0186] Glucocorticoids (adrenocorticoid steroids) are among thecompounds for which delivery across skin can be enhanced by thedelivery-enhancing transporters of the invention. Conjugatedglucocorticoids of the invention are useful for treating inflammatoryskin diseases, for example. Exemplary glucocorticoids include, e.g.,hydrocortisone, prenisone (deltasone) and predrisonlone (hydeltasol).Examples of particular conditions include eczema (including atopicdermatitis, contact dermatitis, allergic dermatitis), bullous disease,collagen vascular diseases, sarcoidosis, Sweet's disease, pyodermagangrenosum, Type I reactive leprosy, capillary hemangiomas, lichenplanus, exfoliative dermatitis, erythema nodosum, hormonal abnormalities(including acne and hirsutism), as well as toxic epidermal necrolysis,erythema multiforme, cutaneous T-cell lymphoma, discoid lupuserythematosus, and the like.

[0187] Retinoids are another example of a biologically active agent forwhich one can use the delivery-enhancing transporters of the inventionto enhance delivery into and across one or more layers of the skin orother epithelial or endothelial tissue. Retinoids that are presently inuse include, for example retinol, tretinoin, isotretinoin, etretinate,acitretin, and arotinoid. Conditions that are treatable using retinoidsconjugated to the delivery-enhancing transporters of the inventioninclude, but are not limited to, acne, keratinization disorders, skincancer, precancerous conditions, psoriasis, cutaneous aging, discoidlupus erythematosus, scleromyxedema, verrucous epidermal nevus,subcomeal pustular dermatosis, Reiter's syndrome, warts, lichen planus,acanthosis nigricans, sarcoidosis, Grover's disease, porokeratosis, andthe like.

[0188] Cytotoxic and immunosuppressive drugs constitute an additionalclass of drugs for which the delivery-enhancing transporters of theinvention are useful. These agents are commonly used to treathyperproliferative diseases such as psoriasis, as well as for immunediseases such as bullous dermatoses and leukocytoclastic vasculitis.Examples of such compounds that one can conjugate to thedelivery-enhancing transporters of the invention include, but are notlimited to, antimetabolites such as methotrexate, azathioprine,fluorouracil, hydroxyurea, 6-thioquanine, mycophenolate, chlorambucil,vinicristine, vinblasrine and dactinomycin. Other examples arealkylating agents such as cyclophosphamide, mechloroethaminehydrochloride, carmustine. taxol, tacrolimus and vinblastine areadditional examples of useful biological agents, as are dapsone andsulfasalazine. Ascomycins, such as Cyclosporine, FK506 (tacrolimus), andrapamycin (e.g., U.S. Pat. No. 5,912,253) and analogs of such compoundsare of particular interest (e.g., Mollinson et al., Current Pharm.Design 4(5):367-380 (1998); U.S. Pat. Nos. 5,612,350; 5,599,927;5,604,294; 5,990,131; 5,561,140; 5,859,031; 5,925,649; 5,994,299;6,004,973 and 5,508,397). Cyclosporins include cyclosporin A, B, C, D ,G and M. See, e.g., U.S. Pat. Nos. 6,007,840; and 6,004,973. Forexample, such compounds are useful in treating psoriasis, eczema(including atopic dermatitis, contact dermatitis, allergic dermatitis)and alopecia areata.

[0189] The delivery-enhancing transporters can be conjugated to agentsthat are useful for treating conditions such as lupus erythematosus(both discoid and systemic), cutaneous dermatomyositis, porphyriacutanea tarda and polymorphous light eruption. Agents useful fortreating such conditions include, for example, quinine, chloroquine,hydroxychloroquine, and quinacrine.

[0190] The delivery-enhancing transporters of the invention are alsouseful for transdermal delivery of antiinfective agents. For example,antibacterial, antifungal and antiviral agents can be conjugated to thedelivery-enhancing transporters. Antibacterial agents are useful fortreating conditions such as acne, cutaneous infections, and the like.Antifungal agents can be used to treat tinea corporis, tinea pedis,onychomycosis, candidiasis, tinea versicolor, and the like. Because ofthe delivery-enhancing properties of the conjugates, these conjugatesare useful for treating both localized and widespread infections.Antifungal agents are also useful for treating onychomycosis. Examplesof antifungal agents include, but are not limited to, azole antifungalssuch as itraconazole, myconazole and fluconazole. Examples of antiviralagents include, but are not limited to, acyclovir, famciclovir, andvalacyclovir. Such agents are useful for treating viral diseases, e.g.,herpes.

[0191] Another example of a biologically active agent for whichenhancement of delivery by conjugation to the delivery-enhancingtransporters of the invention is desirable are the antihistamines. Theseagents are useful for treating conditions such as pruritus due tourticaria, atopic dermatitis, contact dermatitis, psoriasis, and manyothers. Examples of such reagents include, for example, terfenadine,astemizole, lorotadine, cetirizine, acrivastine, temelastine,cimetidine. ranitidine, famotidine, nizatidine, and the like. Tricyclicantidepressants can also be delivered using the delivery-enhancingtransporters of the invention.

[0192] Topical antipsoriasis drugs are also of interest. Agents such ascorticosteroids, calcipotriene, and anthralin can be conjugated to thedelivery-enhancing transporters of the invention and applied to skin.

[0193] The delivery-enhancing transporters of the invention are alsouseful for enhancing delivery of photochemotherapeutic agents into andacross one or more layers of skin and other epithelial tissues. Suchcompounds include, for example, the psoralens, and the like. Sunscreencomponents are also of interest; these include p-aminobenzoic acidesters, cinnamates and salicylates, as well as benzophenones,anthranilates, and avobenzone.

[0194] Pain relief agents and local anesthetics constitute another classof compounds for which conjugation to the delivery-enhancingtransporters of the invention can enhance treatment. Lidocaine,bupibacaine, novocaine, procaine, tetracaine, benzocaine, cocaine, andthe opiates, are among the compounds that one can conjugate to thedelivery-enhancing transporters of the invention.

[0195] Other biological agents of interest include, for example,minoxidil, keratolytic agents, destructive agents such as podophyllin,hydroquinone, capsaicin, masoprocol, colchicine, and gold.

[0196] B. Gastrointestinal Administration

[0197] The delivery-enhancing transporters of the invention are alsouseful for delivery of conjugated drugs by gastrointestinaladministration. Gastrointestinal administration can be used for bothsystemically active drugs, and for drugs that act in thegastrointestinal epithelium.

[0198] Among the gastrointestinal conditions that are treatable usingappropriate reagents conjugated to the delivery-enhancing transportersare Crohn's disease (e.g., cyclosporin and FK506), ulcerative colitis,gastrointestinal ulcers, peptic ulcer disease, imbalance of salt andwater absorption (can lead to constipation, diarrhea, or malnutrition),abnormal proliferative diseases, and the like. Ulcer treatments include,for example, drugs that reduce gastric acid secretion, such as H₂histamine inhibitors (e.g., cymetidine and ranitidine) and inhibitors ofthe proton-potassium ATPase (e.g., lansoprazle amd omeprazle), andantibiotics directed at Helicobacter pylori.

[0199] Antibiotics are among the biologically active agents that areuseful when conjugated to the delivery-enhancing transporters of theinvention, particularly those that act on invasive bacteria, such asShigella, Salmonella, and Yersinia. Such compounds include, for example,norfloxacin, ciprofloxacin, trimethoprim, sulfamethyloxazole, and thelike.

[0200] Anti-neoplastic agents can also be conjugated to thedelivery-enhancing transporters of the invention and administered by thegastrointestinal route. These include, for example, cisplatin,methotrexate, taxol, fluorouracil, mercaptopurine, donorubicin,bleomycin, and the like.

[0201] C. Respiratory Tract Administration

[0202] The delivery-enhancing transporters of the invention can alsoused to enhance administration of drugs through the respiratory tract.The respiratory tract, which includes the nasal mucosa, hypopharynx, andlarge and small airway structures, provides a large mucosal surface fordrug absorption. The enhanced penetration of the conjugated agents intoand across one or more layers of the epithelial tissue that is providedby the delivery-enhancing transporters of the invention results inamplification of the advantages that respiratory tract delivery has overother delivery methods. For example, lower doses of an agent are oftenneeded to obtain a desired effect, a local therapeutic effect can occurmore rapidly, and systemic therapeutic blood levels of the agent areobtained quickly. Rapid onset of pharmacological activity can resultfrom respiratory tract administration. Moreover, respiratory tractadministration generally has relatively few side effects.

[0203] The transporters of the invention can be used to deliverbiological agents that are useful for treatment of pulmonary conditions.Examples of conditions treatable by nasal administration include, forexample, asthma. These compounds include antiinflammatory agents, suchas corticosteroids, cromolyn, and nedocromil, bronchodialators such asβ2-selective adronergic drugs and theophylline, and immunosuppressivedrugs (e.g., cyclosporin and FK506). Other conditions include, forexample, allergic rhinitis (which can be treated with glucocorticoids),and chronic obstructive pulmonary disease (emphysema). Other drugs thatact on the pulmonary tissues and can be delivered using the transportersof the invention include beta-agonists, mast cell stabilizers,antibiotics, antifungal and antiviral agents, surfactants, vasoactivedrugs, sedatives and hormones.

[0204] Respiratory tract administration is useful not only for treatmentof pulmonary conditions, but also for delivery of drugs to distanttarget organs via the circulatory system. A wide variety of such drugsand diagnostic agents can be administered through the respiratory tractafter conjugation to the delivery-enhancing transporters of theinvention.

[0205] D. Delivery of Agents across the Blood Brain Barrier

[0206] The delivery-enhancing transporters are also useful fordelivering biologically active and diagnostic agents across the bloodbrain barrier. The agents are useful for treating ischemia (e.g., usingan anti-apoptotic drug), as well as for delivering neurotransmitters andother agents for treating various conditions such as schizophrenia,Parkinson's disease, pain (e.g., morphine, the opiates). The5-hydroxytryptamine receptor antagonist is useful for treatingconditions such as rmigraine headaches and anxiety.

[0207] E. Diagnostic imaging and contrast agents

[0208] The delivery-enhancing transporters of the invention are alsouseful for delivery of diagnostic imaging and contrast agents into andacross one or more layers of an epithelial and/or endothelial tissue.Examples of diagnostic agents include substances that are labeled withradioactivity, such as ⁹⁹mTc glucoheptonate, or substances used inmagnetic resonance imaging (MRI) procedures such as gadolinium dopedchelation agents (e.g. Gd-DTPA). Other examples of diagnostic agentsinclude marker genes that encode proteins that are readily detectablewhen expressed in a cell (including, but not limited to,(β-galactosidase, green fluorescent protein, luciferase, and the like. Awide variety of labels may be employed, such as radionuclides, fluors,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands(particularly haptens), etc.

[0209] Biologically-Active and Diagnostic Molecules Useful with theDelivery-enhancing Transporters

[0210] The delivery-enhancing transporters can be conjugated to a widevariety of biologically active agents and molecules that have diagnosticuse.

[0211] A. Small Organic Molecules

[0212] Small organic molecule therapeutic agents can be advantageouslyattached to linear polymeric compositions as described herein, tofacilitate or enhance transport across one or more layers of anepithelial or endothelial tissue. For example, delivery of highlycharged agents, such as levodopa (L-3,4-dihydroxy-phenylalanine; L-DOPA)may benefit by linkage to delivery-enhancing transporters as describedherein. Peptoid and peptidomimetic agents are also contemplated (e.g.,Langston (1997) DDT2:255; Giannis et al. (1997) Advances Drug Res.29:1). Also, the invention is advantageous for delivering small organicmolecules that have poor solubilities in aqueous liquids, such as serumand aqueous saline. Thus, compounds whose therapeutic efficacies arelimited by their low solubilities can be administered in greater dosagesaccording to the present invention, and can be more efficacious on amolar basis in conjugate form, relative to the non-conjugate form, dueto higher uptake levels by cells.

[0213] Since a significant portion of the topological surface of a smallmolecule is often involved, and therefore required, for biologicalactivity, the small molecule portion of the conjugate in particularcases may need to be severed from the attached delivery-enhancingtransporter and linker moiety (if any) for the small molecule agent toexert biological activity after crossing the target epithelial tissue.For such situations, the conjugate preferably includes a cleavablelinker for releasing free drug after passing through an epithelialtissue.

[0214]FIG. 5D and FIG. 5E are illustrative of another aspect of theinvention, comprising taxane- and taxoid anticancer conjugates whichhave enhanced trans-epithelial tissue transport rates relative tocorresponding non-conjugated forms. The conjugates are particularlyuseful for inhibiting growth of cancer cells. Taxanes and taxoids arebelieved to manifest their anticancer effects by promotingpolymerization of microtubules (and inhibiting depolymerization) to anextent that is deleterious to cell function, inhibiting cell replicationand ultimately leading to cell death.

[0215] The term “taxane” refers to paclitaxel (FIG. 5F, R′=acetyl,R″=benzyl) also known under the trademark “TAXOL”) and naturallyoccurring, synthetic, or bioengineered analogs having a backbone corethat contains the A, B, C and D rings of paclitaxel, as illustrated inFIG. 5G. FIG. 5F also indicates the structure of “TAXOTERE™” (R′=H,R″=BOC), which is a somewhat more soluble synthetic analog of paclitaxelsold by Rhone-Poulenc. “Taxoid” refers to naturally occurring, syntheticor bioengineered analogs of paclitaxel that contain the basic A, B and Crings of paclitaxel, as shown in FIG. 5H. Substantial synthetic andbiological information is available on syntheses and activities of avariety of taxane and taxoid compounds, as reviewed in Suffness (1995)Taxol: Science and Applications, CRC Press, New York, N.Y., pp. 237-239,particularly in Chapters 12 to 14, as well as in the subsequentpaclitaxel literature. Furthermore, a host of cell lines are availablefor predicting anticancer activities of these compounds against certaincancer types, as described, for example, in Suffness at Chapters 8 and13.

[0216] The delivery-enhancing transporter is conjugated to the taxane ortaxoid moiety via any suitable site of attachment in the taxane ortaxoid. Conveniently, the transport polymer is linked via a C2′-oxygenatom, C7-oxygen atom, using linking strategies as above. Conjugation ofa transport polymer via a C7-oxygen leads to taxane conjugates that haveanticancer and antitumor activity despite conjugation at that position.Accordingly, the linker can be cleavable or non-cleavable. Conjugationvia the C2′-oxygen significantly reduces anticancer activity, so that acleavable linker is preferred for conjugation to this site. Other sitesof attachment can also be used, such as C10.

[0217] It will be appreciated that the taxane and taxoid conjugates ofthe invention have improved water solubility relative to taxol (≈0.25μg/mL) and taxotere (6-7 μg/mL). Therefore, large amounts ofsolubilizing agents such as “CREMOPHOR EL” (polyoxyethylated castoroil), polysorbate 80 (polyoxyethylene sorbitan monooleate, also known as“TWEEN 80”), and ethanol are not required, so that side-effectstypically associated with these solubilizing agents, such asanaphylaxis, dyspnea, hypotension, and flushing, can be reduced.

[0218] B. Metals

[0219] Metals can be transported into and across one or more layers ofepithelial and endothelial tissues using chelating agents such astexaphyrin or diethylene triamine pentacetic acid (DTPA), conjugated toa delivery-enhancing transporter of the invention, as illustrated byExample. These conjugates are useful for delivering metal ions forimaging or therapy. Exemplary metal ions include Eu, Lu, Pr, Gd, Tc99m,Ga67, In111, Y90, Cu67, and Co57. Preliminary membrane-transport studieswith conjugate candidates can be performed using cell-based assays suchas described in the Example section below. For example, using europiumions, cellular uptake can be monitored by time-resolved fluorescencemeasurements. For metal ions that are cytotoxic, uptake can be monitoredby cytotoxicity.

[0220] C. Macromolecules

[0221] The enhanced transport methods of the invention are particularlysuited for enhancing transport into and across one or more layers of anepithelial or endothelial tissue for a number of macromolecules,including, but not limited to proteins, nucleic acids, polysaccharides,and analogs thereof. Exemplary nucleic acids include oligonucleotidesand polynucleotides formed of DNA and RNA, and analogs thereof, whichhave selected sequences designed for hybridization to complementarytargets (e.g., antisense sequences for single- or double-strandedtargets), or for expressing nucleic acid transcripts or proteins encodedby the sequences. Analogs include charged and preferably unchargedbackbone analogs, such as phosphonates (preferably methyl phosphonates),phosphoramidates (N3′ or N5′), thiophosphates, unchargedmorpholino-based polymers, and protein nucleic acids (PNAs). Suchmolecules can be used in a variety of therapeutic regimens, includingenzyme replacement therapy, gene therapy, and anti-sense therapy, forexample.

[0222] By way of example, protein nucleic acids (PNA) are analogs of DNAin which the backbone is structurally homomorphous with a deoxyribosebackbone. The backbone consists of N-(2-aminoethyl)glycine units towhich the nucleobases are attached. PNAs containing all four naturalnucleobases hybridize to complementary oligonucleotides obeyingWatson-Crick base-pairing rules, and is a true DNA mimic in terms ofbase pair recognition (Egholm et al. (1993) Nature 365:566-568. Thebackbone of a PNA is formed by peptide bonds rather than phosphateesters, making it well-suited for anti-sense applications. Since thebackbone is uncharged, PNA/DNA or PNA/RNA duplexes that form exhibitgreater than normal thermal stability. PNAs have the additionaladvantage that they are not recognized by nucleases or proteases. Inaddition, PNAs can be synthesized on an automated peptides synthesizerusing standard t-Boc chemistry. The PNA is then readily linked to atransport polymer of the invention.

[0223] Examples of anti-sense oligonucleotides whose transport into andacross epithelial and endothelial tissues can be enhanced using themethods of the invention are described, for example, in U.S. Pat. No.5,594,122. Such oligonucleotides are targeted to treat humanimmunodeficiency virus (HIV). Conjugation of a transport polymer to ananti-sense oligonucleotide can be effected, for example, by forming anamide linkage between the peptide and the 5′-terminus of theoligonucleotide through a succinate linker, according towell-established methods. The use of PNA conjugates is furtherillustrated in Example 11 of PCT Application PCT/US98/10571. FIG. 7 ofthat application shows results obtained with a conjugate of theinvention containing a PNA sequence for inhibiting secretion ofgamma-interferon (γ-IFN) by T cells, as detailed in Example 11. As canbe seen, the anti-sense PNA conjugate was effective to block γ-IFNsecretion when the conjugate was present at levels above about 10 μM. Incontrast, no inhibition was seen with the sense-PNA conjugate or thenon-conjugated antisense PNA alone.

[0224] Another class of macromolecules that can be transported acrossone or more layers of an epithelial or endothelial tissue is exemplifiedby proteins, and in particular, enzymes. Therapeutic proteins include,but are not limited to replacement enzymes. Therapeutic enzymes include,but are not limited to, alglucerase, for use in treating lysozomalglucocerebrosidase deficiency (Gaucher's disease), alpha-L-iduronidase,for use in treating mucopolysaccharidosis I,alpha-N-acetylglucosamidase, for use in treating sanfilippo B syndrome,lipase, for use in treating pancreatic insufficiency, adenosinedeaminase, for use in treating severe combined immunodeficiencysyndrome, and triose phosphate isomerase, for use in treatingneuromuscular dysfunction associated with triose phosphate isomerasedeficiency.

[0225] In addition, and according to an important aspect of theinvention, protein antigens may be delivered to the cytosoliccompartment of antigen-presenting cells (APCs), where they are degradedinto peptides. The peptides are then transported into the endoplasmicreticulum, where they associate with nascent HLA class I molecules andare displayed on the cell surface. Such “activated” APCs can serve asinducers of class I restricted antigen-specific cytotoxic T-lymphocytes(CTLs), which then proceed to recognize and destroy cells displaying theparticular antigen. APCs that are able to carry out this processinclude, but are not limited to, certain macrophages, B cells anddendritic cells. In one embodiment, the protein antigen is a tumorantigen for eliciting or promoting an immune response against tumorcells. The transport of isolated or soluble proteins into the cytosol ofAPC with subsequent activation of CTL is exceptional, since, with fewexceptions, injection of isolated or soluble proteins does not resulteither in activation of APC or induction of CTLs. Thus, antigens thatare conjugated to the transport enhancing compositions of the presentinvention may serve to stimulate a cellular immune response in vitro orin vivo.

[0226] In another embodiment, the invention is useful for deliveringimmunospecific antibodies or antibody fragments to the cytosol tointerfere with deleterious biological processes such as microbialinfection. Recent experiments have shown that intracellular antibodiescan be effective antiviral agents in plant and mammalian cells (e.g.,Tavladoraki et al. (1993) Nature 366:469; and Shaheen et al. (1996) J.Virol. 70:3392. These methods have typically used single-chain variableregion fragments (scFv), in which the antibody heavy and light chainsare synthesized as a single polypeptide. The variable heavy and lightchains are usually separated by a flexible linker peptide (e.g., of 15amino acids) to yield a 28 kDa molecule that retains the high affinityligand binding site. The principal obstacle to wide application of thistechnology has been efficiency of uptake into infected cells. But byattaching transport polymers to scFv fragments, the degree of cellularuptake can be increased, allowing the immunospecific fragments to bindand disable important microbial components, such as HIV Rev, HIV reversetranscriptase, and integrase proteins.

[0227] D. Peptides

[0228] Peptides to be delivered by the enhanced transport methodsdescribed herein include, but should not be limited to, effectorpolypeptides, receptor fragments, and the like. Examples includepeptides having phosphorylation sites used by proteins mediatingintra-cellular signals. Examples of such proteins include, but are notlimited to, protein kinase C, RAF-1, p21Ras, NF-κB, C-JUN, andcytoplasmic tails of membrane receptors such as IL-4 receptor, CD28,CTLA-4, V7, and MHC Class I and Class II antigens.

[0229] When the delivery-enhancing transporter is also a peptide,synthesis can be achieved either using an automated peptide synthesizeror by recombinant methods in which a polynucleotide encoding a fusionpeptide is produced, as mentioned above.

EXAMPLES

[0230] The following examples are offered to illustrate, but not tolimit the present invention.

Example 1 Penetration of Biotinylated Polymers of D-arginine into theSkin of Nude Mice

[0231] This Example demonstrates that poly-arginine heptamers candeliver conjugated biotin into and across layers of the skin, bothfollicularly and interfollicularly, and into the dermis.

[0232] Methods

[0233] Biotinylated peptides were synthesized using solid phasetechniques and commercially available Fmoc amino acids, resins, andreagents (PE Biosystems, Foster City Calif., and Bachem Torrence,Calif.) on a Applied Biosystems 433 peptide synthesizer. Fastmoc cycleswere used with O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexfluorophosphate (HATU) substituted for HBTU/HOBt as the couplingreagent. Prior to the addition of biotin to the amino terminus of thepeptide, amino caproic acid (aca) was conjugated and acted as a spacer.The peptides were cleaved from the resin using 96% trifluoroacetic acid,2% triisopropyl silane, and 2% phenol for between 1 and 12 hours. Thelonger reaction times were necessary to completely remove the Pbfprotecting groups from the polymers of arginine. The peptidessubsequently were filtered from the resin, precipitated using diethylether, purified using HPLC reverse phase columns (Alltech Altima,Chicago, Ill.) and characterized using either electrospray or matrixassisted laser desorption mass spectrometry (Perceptive Biosystems,Boston, Mass.).

[0234] Varying concentrations (1 mM-10 μM) of a heptamer of D-argininewith biotin covalently attached to the amino terminus using an aminocaproic acid spacer (bio r7), dissolved in phosphate buffered saline(PBS), were applied to the back of anesthetized nude mice. Samples (100μl) were applied as a liquid without excipient, prevented fromdispersing by a Vaseline™ barrier, and allowed to penetrate for fifteenminutes. At the end of this period the animal was sacrificed, therelevant sections of skin were excised, embedded in mounting medium(OCT), and frozen. Frozen sections (5 microns) were cut using acryostat, collected on slides, and stained with fluorescently labeledstreptavidin (Vector Laboratories, Burlingame, Calif.). The slides werefixed in acetone at 4° C. for ten minutes, air dried, soaked in PBS forfive minutes, blocked with normal goat serun for five minutes, andwashed with PBS for five minutes. The section was stained by incubationwith fluorescently labeled streptavidin at 30 μg/ml for thirty minutes,washed with PBS, counterstained with propidium iodide (1 μg/ml) for twominutes, and the section was mounted with Vectashield™ mounting media.Slides were analyzed by fluorescent microscopy. Parallel studies weredone using streptavidin-horse radish peroxidase rather thanfluorescein-streptavidin. The biotinylated peptide was visualized bytreatment of the sections with the horseradish peroxidase substratediaminobenzadine, and visualization with light microscopy.

[0235] Results

[0236] Biotinylated arginine heptamer crossed into and across theepidermis and into the dermis. The cytosol and nuclei of all cells inthe field were fluorescent, indicating penetration into virtually everycell of the nude mouse skin in the section. The staining pattern wasconsistent with unanticipated transport that was both follicular andinterfollicular. In addition, positive cells were apparent in papillaryand reticular dermis. In contrast, no staining was apparent in micetreated with biotin alone, or phosphate buffered saline alone.

Example 2 Penetration of Biotinylated Polymers of D-arginine into theSkin of Normal Balb/C Mice

[0237] Varying concentrations (1 mM-100 μM) of a heptamer of D-argininewith biotin covalently attached to the amino terminus using an aminocaproic acid spacer (bio r7), dissolved in PBS, were applied to a skinof the groin of an anesthetized Balb/C mice. Sample (100 μl) was appliedas a liquid within excipient and prevented from dispersing by aVaseline™ barrier and allowed to penetrate for thirty minutes. At theend of this period animal was sacrificed, the relevant section of skinwas excised, embedded in mounting medium (OCT) and frozen. Frozensections were cut using a cryostat, collected on slides, and stainedwith fluorescently labeled streptavidin (Vector Laboratories,Burlingame, Calif.) as described in Example 1. Slides were analyzed byfluorescent microscopy.

[0238] Results

[0239] As with the skin from nude mice, biotinylated arginine heptamercrossed into and across the epidermis and into the dermis. The cytosoland nuclei of all cells in the field were fluorescent, indicatingpenetration into virtually every cell of the nude mouse skin in thesection. The staining pattern was consistent with unanticipatedtransport that was both follicular and interfollicular. In addition,positive cells were apparent in papillary and reticular dermis. Incontrast, no staining was apparent in mice treated with biotin alone, orphosphate buffered saline alone.

Example 3 Penetration of Biotinylated Polymers of D-arginine into NormalHuman Skin Grafted onto Nude Mice

[0240] Varying concentrations (1 mM-100 μM) of a heptamer of D-argininewith biotin covalently attached to the amino terminus using an aminocaproic acid spacer (bio r7), dissolved in PBS, were applied to humanforeskin grafts on the back of SCID mice (see, e.g., Deng et al. (1997)Nature Biotechnol. 15: 1388-1391; Khavari et al. (1997) Adv. Clin. Res.15:27-35; Choate and Khavari (1997) Human Gene Therapy 8:895-901).Samples (100 μl) were applied as a liquid within excipient and preventedfrom dispersing by a Vaseline™ barrier and allowed to penetrate forfifteen minutes. At the end of this period animal was sacrificed, therelevant section of skin was excised, embedded in mounting medium (OCT)and frozen. Frozen sections were cut using a cryostat, collected onslides, and stained with fluorescently labeled streptavidin (VectorLaboratories, Burlingame, Calif.) as described in Example 1. Slides wereanalyzed by fluorescent microscopy.

[0241] Results

[0242] As with the skin from nude and normal mice, biotinylated arginineheptamer crossed into and across the epidermis and into the dermis ofthe human skin. The cytosol and nuclei of all cells in the field werefluorescent, indicating penetration into and through the epidermis anddermis. Intense staining was seen at both 20× and 40×magnification. Thestaining pattern was consistent with unanticipated transport that wasboth follicular and interfollicular. In addition, positive cells wereapparent in papillary and reticular dermis. In contrast, no staining wasapparent in mice treated with biotin alone, or phosphate buffered salinealone, and very little staining was observed with the biotinylatedarginine pentamer conjugate, either at low or high magnification.

Example 4 Increased Penetration of Biotinylated Polymers of D-arginineinto Skin of Nude Mouse Using Plastic Wrap or a Lotion Excipient

[0243] Varying concentrations (1 mM-100 μM) of a heptamer of D-argininewith biotin covalently attached to the amino terminus using an aminocaproic acid spacer (bio r7), dissolved in PBS, and mixed with an equalvolume of Lubriderm™. The lotion mixture was then applied to the back ofnude mice and allowed to penetrate for thirty, sixty, and 120 minutes.Alternatively, sample (100 μl) was applied as a liquid without excipientand prevented from evaporating by wrapping plastic wrap over the samplesealed with Vaseline™. The samples were allowed to penetrate for thirty,sixty, and 120 minutes. At the end of this period animal was sacrificed,the relevant section of skin was excised, embedded in mounting medium(OCT) and frozen. Frozen sections were cut using a cryostat, collectedon slides, and stained with fluorescently labeled streptavidin (VectorLaboratories, Burlingame, Calif.) as described in Example 1. Slides wereanalyzed by fluorescent microscopy.

[0244] Results

[0245] Both lotion and plastic wrap resulted in increased uptakecompared with staining without excipient. Lotion was more effective thanplastic wrap in enhancing uptake of the conjugate. Biotinylated argininepentamers crossed into and across several skin layers, reaching both thecytosol and nuclei of epidermal cell layers, both follicular andinterfollicular. In addition, positive cells were apparent in papillaryand reticular dermis.

Example 5 Penetration of Cyclosporin Conjugated to a BiotinylatedPentamer, Heptamer, and Nonamer of D-arginine into the Skin of Nude Mice

[0246] Methods

[0247] A. Linking Cyclosporin to Delivery-enhancing Transporters

[0248] 1. Preparation of the α-chloroacetyl Cyclosporin A derivative.

[0249] The α-chloroacetyl cyclosporin A derivative was prepared as shownin FIG. 1. Cyclosporin A (152.7 mg, 127 μmol) and chloroacetic acidanhydride (221.7 mg; 1300 μmol) were placed into a dry flask underN₂-atmosphere. Pyridine (1.0 mL) was added and the solution was heatedto 50° C. (oil bath). After 16 hours the reaction was cooled to roomtemperature and quenched with water (4.0 mL). The resulting suspensionwas extracted with diethylether (Σ15 mL). The combined organic layerswere dried over MgSO₄. Filtration and evaporation of solvents in vacuodelivered a yellow oil, which was purified by flash chromatography onsilica gel (eluent: EtOAc/hexanes: 40%-80%) yielding 136 mg (106.4 μmol,83%) of the desired product.

[0250] 2. Coupling to Transporter molecules

[0251] A general procedure for the coupling of cysteine containingpeptides to the α-chloro acetyl Cyclosporin A derivative is shown inFIG. 2.

[0252] a. Labeled Peptides

[0253] The cyclosporin A derivative and the labeled peptide (1equivalent) were dissolved in DMF (˜10 mmol of Cyclosporin Aderivative/mL DMF) under an N₂-atmosphere. Diisopropylethylamine (10equivalents) was added and stirring at room temperature was continueduntil all starting material was consumed (usually after 16 hours) (FIG.3). The solvents were removed in vacuo and the crude reaction productwas dissolved in water and purified by reversed phase high pressureliquid chromatography (RP-HPLC) (eluent: water/MeCN *TFA). The productswere obtained in the following yields:

[0254] B-aca-r5-Ala-Ala-Cys-O-acyl-Cyclosporin A: 47%

[0255] B-aca-r7-Cys-O-acyl-Cyclosporin A: 43%

[0256] B-aca-r9-Cys-O-acyl-Cyclosporin A: 34%

[0257] B-aca-Cys-O-acyl-Cyclosporin A: 55%

[0258] b. Unlabeled Peptides

[0259] The peptide (34.7 mg, 15.3 μmol) and the Cyclosporin A derivative(19.6 mg, 15.3 μmol) were dissolved in DMF (1.0 mL) under anN₂-atmosphere (FIG. 4). Disopropylethylamine (19.7 mg, 153 μmol) wasadded and stirring at room temperature was continued. After 12 hours thesolvent was removed in vacuo. The crude material was dissolved in waterand purified by RP-HPLC (eluent: water/MeCN *TFA) yielding the pureproduct (24.1 mg, 6.8 mmol, 44%).

[0260] B. Analysis of Transport Across Skin

[0261] Varying concentrations (1 mM-100 μM) of cyclosporin conjugated toeither biotinylated pentamer, heptamer, or nonamers of D-arginine (bior5, r7, or r9), dissolved in PBS, were applied to the back of nude mice.Samples (100 μl ) were applied as a liquid within excipient andprevented from dispersing by a Vaseline™ barrier and allowed topenetrate for thirty, sixty, and 120 minutes. At the end of this periodanimal was sacrificed, the relevant section of skin was excised,embedded in mounting medium (OCT) and frozen. Frozen sections were cutusing a cryostat, collected on slides, and stained with fluorescentlylabeled streptavidin (Vector Laboratories, Burlingame, Calif.) asdescribed in Example 1. Slides were analyzed by fluorescent microscopy

[0262] Results

[0263] The conjugates of cyclosporin with biotinylated heptamers andnonamers of D-arginine effectively entered into and across the epidermisand into the dermis of the skin of nude mice. In contrast, very littleuptake was seen using a conjugate between a pentamer of arginine andcyclosporin, and no staining was seen with a PBS control. The cytosoland nuclei of all cells in the field were fluorescent, indicatingpenetration into and through the epidermis and dermis. The stainingpattern was consistent with unanticipated transport that was bothfollicular and interfollicular. In addition, positive cells wereapparent in papillary and reticular dermis. These results demonstrateremarkable uptake only when sufficient guanidinyl groups are included inthe delivery-enhancing transporter.

Example 6 Demonstration That a D-arginine Heptamer Can Penetrate HumanSkin

[0264] Human and murine skin differ significantly in a number of ways,with human epidermis being considerably thicker. To determine if theD-arginine heptamers/cyclosporin A (r7 CsA) conjugate could alsopenetrate human skin, biotin r7 CsA was applied to full thickness humanskin grafted onto the back of a SCID mouse. As in murine skin,conjugated cyclosporin A penetrated human epidermis and dermis.Fluorescence was observed in both the cytosol and the nuclei of cells intissue exposed to biotinylated peptides alone, but in sections stainedwith biotin r7 CsA the majority of fluorescence was cytosolic,consistent with r7 CsA binding to cyclosporin A's known cytoplasmictargets.

Example 7 Demonstration That Cyclosporin A-transporter Conjugates EnterT Cells in the Dermis

[0265] Methods

[0266] Inhibition of IL-2 secretion by releasable R7-CsA conjugate.Jurkat cells (5×10⁴) were incubated with varying concentrations of anonreleasable or releasable R7-CsA conjugate or CsA overnight at 37° C.to allow for the release of the active form of CsA prior to stimulationwith PMA and ionomycin. T cells subsequently were stimulated to produceIL-2 by addition of 10 ng/ml PMA (Sigma, St. Louis, Mo.) and 1 μMionomycin (CalBiochem, San Diego, Calif.). Cultures were incubatedovernight at 37° C. and supematants were collected and IL-2 was measuredusing a fluorescent ELISA. Briefly, plates were coated with 4 μg/mlanti-human IL-2 antibody (BD Pharmingen, San Diego, Calif.), blockedwith PBS containing 10% FBS for 1 hour at room temperature, washed, andsupernatants added and incubated for 1 hour. Media was removed andbiotinylated anti-human IL-2 (1.6 μg/ml), was added for one hour. Theplates were washed, and then europium labeled streptavidin (0.04 ng/ml)was added for one hour. After another wash, enhancement solution wasadded and the resulting fluorescence was measured using a Wallac platereader (Wallac, Turku, Finland).

[0267] Results

[0268] To determine whether biotinylated D-arginine heptamer-cyclosporin(r7 CsA) conjugate would reach infiltrating T cells within inflamed skinin vivo, biotin r7 CsA was applied to the site of inflammation on theback of a mouse with experimentally induced contact dermatitis. Inflamedskin was stained with rhodamine labeled goat anti-mouse CD3 to localizeT-cells and with fluorescein labeled streptavidin to localize the biotinr7 CsA. Biotin r7 CsA was found in all CD3[+] T cells in the tissue inaddition to a variety of other cells that probably represent otherinflammatory cells as well as resident fibroblasts. These data indicatethat biotin r7 CsA penetrates inflamed skin to reach key target Tlymphocytes.

Example 8 Synthesis, in vitro and in vivo Activity of a ReleasableConjugate of a Short Oligomer of Arginine and CsA

[0269] Modification of the 2° alcohol of Cyclosporin A results insignificant loss of its biological activity. See, e.g., R. E.Handschumacher, et al., Science 226, 544-7 (1984). Consequently, toensure release of free Cyclosporin A from its conjugate after transportinto cells, Cyclosporin A was conjugated to an oligo-argininetransporter through a pH sensitive linker as shown in FIG. 10. Theresultant conjugate is stable at acidic pH but at pH>7 it undergoes anintramolecular cyclization involving addition of the free amine to thecarbonyl adjacent to Cyclosporin A (FIG. 6), which results in therelease of unmodified Cyclosporin A.

[0270] Another modification in the design of the releasable conjugatewas the use of L-arginine (R), and not D-arginine (r) in thetransporter. While the oligo-D-arginine transporters were used for thehistological experiments to ensure maximal stability of the conjugateand therefore accuracy in determining its location through fluorescence,oligomers of L-arginine were incorporated into the design of thereleasable conjugate to minimize its biological half-life. Consistentwith its design, the resultant releasable conjugate was shown to bestable at acidic pH, but labile at physiological pH in the absence ofserum. This releasable Cyclosporin A conjugate's half-life in pH 7.4 PBSwas 90 minutes.

[0271] Results

[0272] The releasable guanidino-heptamer conjugate of Cyclosporin A wasshown to be biologically active by inhibiting IL-2 secretion by thehuman T cell line, Jurkat, stimulated with PMA and ionomycin in vitro.See R. Wiskocil, et al., J. Immunol 134, 1599-603 (1985). The conjugatewas added 12 hours prior to the addition of PMA/ionomycin and dosedependent inhibition was observed by the releasable R7 CsA conjugate.This inhibition was not observed with a nonreleasable analog (FIG. 6)that differed from the releasable conjugate by retention of the t-Bocprotecting group, which prevented cyclization and resultant release ofthe active drug. The EC₅₀ of the releasable R7 cyclosporin conjugate wasapproximately two fold higher than CsA dissolved in alcohol and added atthe same time as the releasable conjugate.

[0273] The releasable R7 CsA conjugate was assayed in vivo forfunctional activity using a murine model of contact dermatitis.Treatment with the 1% releasable R7 CSA conjugate resulted in 73.9%±4.0reduction in ear inflammation (FIG. 7). No reduction in inflammation wasseen in the untreated ear, indicating that the effect seen in thetreated ear was local and not systemic. Less inhibition was observed inthe ears of mice treated with 0.1 and 0.01% R7-CsA (64.8%±4.0 and40.9%±3.3 respectively), demonstrating that the effect was titratable.Treatment with the fluorinated corticosteroid positive control resultedin reduction in ear swelling (34.1%±6.3), but significantly less thanthat observed for 0.1% releasable R7 CsA (FIG. 7). No reduction ofinflammation was observed in any of the mice treated with unmodifiedCyclosporin A, vehicle alone, R7, or nonreleasable R7 CsA.

Example 9 The Penetration of Copper and Gadolinium-DTPA-r7 Complexesinto the Skin of Nude Mice

[0274] Methods

[0275] 1. Preparation of Metal Complexes

[0276] Step 1-Preparation of Copper-diethylenetriaminepentaacetic AcidComplex (Cu-DTPA)

[0277] Copper carbonate (10 mmol) and diethylenetriaminpentacetic acid(10 mmol) were dissolved in water (150 mL) (FIG. 8). After 18 h, thesolution was centrifuged to removed any solids. The blue solution wasdecanted and lyophilized to provide a blue powder (yields>90%).

[0278] Step 2-Preparation of DTPA Transporter

[0279] The Cu-DTPA was linked to a transporter through an aminocaproicacid spacer using a PE Applied Biosystems Peptide Synthesizer (ABI 433A)(FIG. 9). The material was cleaved from the resin by treatment withtrifluoroacetic acid (TFA) (40 mL), triisopropyl silane (100 μL) andphenol (100 μL) for 18 h. The resin was filtered off and the peptide wasprecipitated by addition of diethyl ether (80 mL). The solution wascentrifliged and the solvent decanted off. The crude solid was purifiedby reverse-phase HPLC using a water/acetonitrile gradient. Treatmentwith TFA resulted in loss of Cu²⁺ ion which needed to be reinserted.

[0280] DTPA-aca-R7-CO2H (10 mg, 0.0063 mmol) and copper sulfate (1.6 mg,0.0063 mmol) were dissolved in water (1 mL). Let gently stir for 18 hand lyophilized to provide product as a white powder (10 mg).

[0281] 2. Analysis of Transport Across Skin

[0282] Metal diethylenetriaminepentaacetic acid (DTPA) complexes wereformed by mixing equimolar amounts of metal salts with DTPA in water for18 hours. At the end of this time, the solutions were centrifuged,frozen and lyophilized. The dried powder was characterized by massspectrometry and used in solid phase peptide synthesis. The metal-DTPAcomplexes were attached to polymers of D- or L-arginine that were stillattached to solid-phase resin used in peptide synthesis. The metal-DTPAcomplexes were attached using an aminocaproic acid spacer. The solidphase peptide synthesis techniques were described in Example 1, with theexception that cleavage of the peptide-DTPA-metal complex intrifluoroacetic acid released the metal. The metal is replaced afterHPLC purification and lyophilization of the peptide-DTPA complex.Replacement of the metal involved incubation of equimolar amounts of themetal salt with the peptide-aminocaproic acid-DTPA complex andsubsequent lyophilization.

[0283] Varying concentrations (1 μM to 1 mM) of the Cu-DTPA-aca-r7complex were applied to the abdominal region of nude mice for 15, 30 and45 minutes. As controls, an equimolar amount of the Cu-DTPA complex wasspotted onto the abdominal region. At the end of the incubation period,the samples were simply wiped off and intense blue color was apparent onthe skin where the Cu-DTPA-aca-r7 complex was spotted and not where theCu-DTPA alone was spotted. In the case of the application of 1 mM,visible blue dye was seen for three days, decreasing with time, butbeing apparent for the full period.

[0284] Varying concentrations (1 μM to 1 mM) of the Gd-DTPA-aca-r7complex are injected into the tail vein of BALB/c mice in 100 μl.Distribution of the Gd is observed in real time using magnetic resonanceimaging. Distribution of the dye is apparent throughout the bloodstream,entering liver, spleen, kidney, and heart. When injected into thecarotid artery of rabbits, the dye is seen to cross the blood brainbarrier.

Example 10 Penetration of Hydrocortisone Conjugated to a BiotinylatedPentamer, Heptamer, and Nonamer of D-arginine into the Skin of Nude Mice

[0285] Methods

[0286] A. Linking of Hydrocortisone to Delivery-enhancing Transporters

[0287] Step 1-Acylation of Hydrocortisone with Chloroacetic Anhydride.

[0288] A solution of hydrocortisone (200 mg, 0.55 mmol) and chloroaceticanhydride (113 mg, 0.66 mmol) in pyridine (5 mL) was stirred at roomtemperature for 2 h (FIG. 10). The solvent was evaporated off and thecrude product was chromatographed on silica using 50% hexanes/ethylacetate as the eluent. Product isolated a whites solid (139 mg, 58%).

[0289] Step 2-Linking to Transporter

[0290] A solution of the chloroacetic ester of hydrocortisone (0.0137mmol), a transporter containing a cysteine residue (0.0137) anddiisopropylethylamine (DIEA) (0.0274 mmol) in dimethylformamide (DMF) (1mL) was stirred at room temperature for 18 h (FIG. 11). The material waspurified via reverse-phase HPLC using a water/acetonitrile gradient andlyophilized to provide a white powder.

[0291] r5 conjugate-12 mg obtained (29% isolated yield)

[0292] r7 conjugate-22 mg obtained (55% isolated yield)

[0293] R7 conjugate-13 mg obtained (33% isolated yield)

[0294] B. Analysis of Transport Across Skin

[0295] Varying concentrations (1 mM-100 μM) of hydrocortisone conjugatedto either biotinylated pentamer, heptamer, or nonamers of D-arginine(bio r5, r7, or r9), dissolved in PBS, were applied to the back of nudemice. Samples (100 μl) were applied as a liquid within excipient andprevented from dispersing by a Vaseline™ barrier and allowed topenetrate for thirty, sixty, and 120 minutes. At the end of this periodanimal was sacrificed, the relevant section of skin was excised,embedded in mounting medium (OCT) and frozen. Frozen sections were cutusing a cryostat, collected on slides, and stained with fluorescentlylabeled streptavidin (Vector Laboratories, Burlingame, Calif.) asdescribed in Example 1. Slides were analyzed by fluorescent microscopy.

[0296] Results

[0297] The conjugates of hydrocortisone with biotinylated heptamers ofD-arginine effectively entered into and across the epidermis and intothe dermis of the skin of nude mice. In contrast, very little uptake wasseen using a conjugate between a pentamer of arginine andhydrocortisone, and no staining was seen with a PBS control. The cytosoland nuclei of all cells in the field were fluorescent, indicatingpenetration into and through the epidermis and dermis. The stainingpattern was consistent with unanticipated transport that was bothfollicular and interfollicular. In addition, positive cells wereapparent in papillary and reticular dermis. These results demonstrateremarkable uptake only when sufficient guanidinyl groups are included inthe delivery-enhancing transporter.

Example 11 Penetration of Taxol Conjugated to a Biotinylated Pentamer,Heptamer, and Nonamer of D-arginine into the Skin of Nude Mice

[0298] Methods

[0299] 1. Conjugation of C-2′ Activated Taxol Derivatives toBiotin-labeled Peptides

[0300] Synthesis of C-2′ Derivatives

[0301] Taxol (48.7 mg, 57.1 μmol) was dissolved in CH₂Cl₂ (3.0 mL) underan N₂-atmosphere. The solution was cooled to 0° C. A stock solution ofthe chloroformate of benzyl-(p-hydroxy benzoate) (200 mmol, in 2.0 mLCH₂Cl₂—freshly prepared from benzyl-(p-hydroxy benzoate) and diphosgene)was added at 0° C. and stirring at that temperature was continued for 5hours, after which the solution was warmed to room temperature (FIG.12). Stirring was continued for additional 10 hours. The solvents wereremoved in vacuo and the crude material was purified by flashchromatography on silica gel (eluent: EtOAc/hexanes 30%-70%) yieldingthe desired taxol C-2′ carbonate (36.3 mg, 32.8 μmol, 57.4%).

[0302] Coupling to biotin-labeled peptides.

[0303] A procedure for coupling to biotin-labeled peptides is shown inFIG. 13. The taxol derivative and the biotin labeled peptide (1.2equivalents) were dissolved in DMF (˜10 μmol/mL DMF) under anN₂-atmosphere. Stock solutions of diisopropylethylamine (1.2 equivalentsin DMF) and DMAP (0.3 equivalents in DMF) were added and stirring atroom temperature was continued until all starting material was consumed.After 16 hours the solvent was removed in vacuo. The crude reactionmixture was dissolved in water and purified by RP-HPLC (eluent:water/MeCN*TFA) yielding the conjugates in the indicated yields:

[0304] B-aca-r5-K-taxol: 3.6 mg, 1.32 mmol, 20%.

[0305] B-aca-r7-K-taxol: 9.8 mg, 3.01 mmol, 44%.

[0306] B-aca-r9-K-taxol: 19.4 mg, 5.1 mmol, 67%.

[0307] Unlabeled C-2′ carbamates:

[0308] The taxol derivative (12.4 mg, 11.2 μmol) and the unlabeledpeptide (27.1 mg, 13.4 μmol) were dissolved in DMF (1.5 mL) under anN₂-atmosphere (FIG. 14). Diisopropylethylamine (1.7 mg, 13.4 μmol) wasadded as a stock solution in DMF, followed by DMAP (0.68 mg, 5.6 μmol)as a stock solution in DMF. Stirring at room temperature was continueduntil all starting material was consumed. After 16 hours the solvent wasremoved in vacuo. The crude material was dissolved in water and purifiedby RP-HPLC (eluent: water/MeCN*TFA) yielding the desired product (16.5mg, 5.9 μmol, 53%).

[0309] Other C-2′ Conjugates

[0310] The taxol derivative (8.7 mg, 7.85 μmol) was dissolved in EtOAc(2.0 mL). Pd/C (10%, 4.0 mg) was added and the reaction flask was purgedwith H₂ five times (FIG. 15A). Stirring under an atmosphere of hydrogenwas continued for 7 hours. The Pd/C was filtered and the solvent wasremoved in vacuo. The crude material (6.7 mg, 6.58 μmol, 84%) obtainedin this way was pure and was used in the next step without furtherpurification.

[0311] The free acid taxol derivative (18.0 mg, 17.7 μmol) was dissolvedin CH₂Cl₂ (2.0 mL). Dicyclohexylcarbodiimide (4.3 mg, 21.3 μmol) wasadded as a stock solution in CH₂Cl₂ (0.1 mL). N-Hydroxysuccinimide (2.0mg, 17.7 μmol) was added as a stock solution in DMF (0.1 mL) (FIG. 15B).Stirring at room temperature was continued for 14 hours. The solvent wasremoved in vacuo and the resultant crude material was purified by flashchromatography on silica gel (eluent: EtOAc/hexanes 40%-80%) yieldingthe desired product (13.6 mg, 12.2 μmol, 69%).

[0312] The activated taxol derivative (14.0 mg, 12.6 μmol) and thepeptide (30.6 mg, 15.1 μmol) were dissolved in DMF (3.0 mL) under anN₂-atmosphere (FIG. 15C). Diisopropylethylamine (1.94 mg, 15.1 μmol) wasadded as a stock solution in DMF (0.1 mL), followed by DMAP (0.76 mg,6.3 μmol) as a stock solution in DMF 0.1 mL). Stirring at roomtemperature was continued until all the starting material was consumed.After 20 hours the solvent was removed in vacuo. The crude material wasdissolved in water and purified by RP-HPLC (eluent: water/MeCN*TFA)yielding the two depicted taxol conjugates in a ration of 1:6 (carbonatevs carbamate, respectively).

[0313] 2. Analysis of Transport Across Skin

[0314] Varying concentrations (1 mM-100 μM) of taxol conjugated toeither biotinylated pentamer, heptamer, or nonamers of D-arginine (bior5, r7, or r9), dissolved in PBS, were applied to the back of nude mice.Samples (100 μl) were applied as a liquid within excipient and preventedfrom dispersing by a Vaseline™ barrier and allowed to penetrate forthirty, sixty, and 120 minutes. At the end of this period animal wassacrificed, the relevant section of skin was excised, embedded inmounting medium (OCT) and frozen. Frozen sections were cut using acryostat, collected on slides, and stained with fluorescently labeledstreptavidin (Vector Laboratories, Burlingame, Calif.) as described inExample 1. Slides were analyzed by fluorescent microscopy.

[0315] Results

[0316] The conjugates of taxol with biotinylated heptamers and nonamersof D-arginine effectively entered into and across the epidermis and intothe dermis of the skin of nude mice. In contrast, very little uptake wasseen using a conjugate between a pentamer of arginine and taxol, and nostaining was seen with a PBS control. The cytosol and nuclei of allcells in the field were fluorescent, indicating penetration into andthrough the epidermis and dermis. The staining pattern was consistentwith unanticipated transport that was both follicular andinterfollicular. In addition, positive cells were apparent in papillaryand reticular dermis. These results demonstrate remarkable uptake onlywhen sufficient guanidinyl groups are included in the delivery-enhancingtransporter.

Example 12 Conjugate of Taxol and Delivery-enhancing Transporter withpH-Releasable Linker

[0317] This Example demonstrates the use of a general strategy forsynthesizing prodrugs that have a delivery-enhancing transporter linkedto a drug by a linker that releases the drug from the delivery-enhancingtransporter upon exposure to physiological pH. In general, a suitablesite on the drug is derivatized to carry an α-chloroacetyl residue.Next, the chlorine is displaced with the thiol of a cysteine residuethat carries an unprotected amine. This scheme is shown in FIG. 16.

[0318] Methods

[0319] Synthesis of Taxol-2′-chloroacetyl

[0320] Taxol (89.5 mg, 104.9 μmol) was dissolved in CH₂Cl₂ (3.5 mL). Thesolution was cooled to 0° C. under an N₂-atmosphere. α-Chloroaceticanhydride (19.7 mg, 115.4 μmol) was added, followed by DIEA (14.8 mg,115.4 μmol). The solution was allowed to warm to room temperature. Afterthin layer chromatography (tlc) analysis indicated complete consumptionof starting material, the solvent was removed in vacuo and the crudematerial was purified by flash chromatography on silica gel (eluent:EtOAC/Hex 20% -50%) yielding the desired material (99.8 mg,quantitative) (FIG. 17).

[0321]¹H-NMR (CDCl₃): δ=8.13 (d, J=7.57 Hz, 2H), 7.72 (d, J=7.57 Hz,2H), 7.62-7.40 (m, 11H), 6.93 (d, J=9.14 Hz, 1H), 6.29-6.23 (m, 2H),6.01 (d, J=7.14 Hz, 1H), 5.66 (d, J=6.80 Hz, 1H), 5.55 (d, J=2.24 Hz,1H), 4.96 (d, J=8.79 Hz, 1H), 4.43 (m, 1H), 4.30 (d, J=8.29 Hz, 1H),4.20-4.15 (m, 2H), 3.81 (d, J=6.71 Hz, 1H), 2.56-2.34 (m, 3H), 2.45 (s,3H), 2.21 (s, 3H), 2.19 (m, 1H), 1.95-1.82 (m, 3H), 1.92 s, (3H), 1.67(s, 3H), 1.22 (s, 3H), 1.13 (s, 3H) ppm.

[0322]¹³C-NMR (CDCl₃): δ=203.6, 171.1, 169.7, 167.3, 167.0, 166.9,166.3, 142.3, 136.4, 133.6, 133.5, 132.9, 132.0, 130.1, 129.2, 121.1,128.7, 128.6, 127.0, 126.5, 84.3, 81.0, 79.0, 76.3, 75.4, 75.2, 75.0,72.2, 72.0, 58.4, 52.7, 45.5, 43.1, 40.1, 35.5, 26.7, 22.6, 22.0, 20.7,14.7, 9.5 ppm.

[0323] Linkage of Taxol to Delivery-Enhancing Transporter

[0324] The peptide (47.6 mg, 22.4 μmol) was dissolved in DMF (1.0 mL)under an N₂-atmosphere. DIEA (2.8 mg, 22.4 μmol) was added. A solutionof taxol-2′-chloroacetate (20.8 mg, 22.4 μmol) in DMF (1.0 mL) wasadded. Stirring at room temperature was continued for 6 hours. Watercontaining 0.1% TFA (1.0 mL) was added, the sample was frozen and thesolvents were lyophilized. The crude material was purified by RP-HPLC(eluent: water/MeCN*0.1% TFA: 85%-15%). A schematic of this reaction isshown in FIG. 18.

[0325] Synthesis of Related Conjugates

[0326] Using the conjugation conditions outlined above, the threeadditional conjugates shown in were synthesized.

[0327] Cytotoxicity Assay

[0328] The taxol conjugates were tested for cytotoxicity in a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT) dyereduction. Results, which are shown in FIG. 20, demonstrate that thetaxol conjugated to r7 with a readily pH-releasable linker (CG 1062;R=Ac in the structure shown in FIG. 19) is significantly more cytotoxicthan either taxol alone or taxol conjugated to r7 with a less-readilypH-releasable linker (CG 1040; R=H in the structure shown in FIG. 19).

Example 13 Structure-Function Relationships of Fluorescently-LabeledPeptides Derived from Tat₄₉₋₅₇

[0329] Methods

[0330] General. Rink amide resin and Boc₂O were purchased fromNovabiochem. Diisopropylcarbodiimide, bromoacetic acid, fluoresceinisothiocyanate (FITC-NCS), ethylenediamine, 1,3-diaminopropane,1,4-diaminobutane, 1,6-diaminohexane, trans-1,6-diaminocyclohexane, andpyrazole-1-carboxamidine were all purchased from Aldrich®. All solventsand other reagents were purchased from commercial sources and usedwithout further purification. The mono-Boc amines were synthesized fromthe commercially available diamines using a literature procedure (10equiv. of diamine and 1 equiv. of Boc₂O in chloroform followed by anaqueous work up to remove unreacted diamine) (34).

[0331] N-tert-butoxycarbonyl-1,6-trans-diaminocyclohexane. Mp 159-161°C.; ¹H NMR (CDCl₃) δ4.35 (br s, 1H), 3.37 (br s, 1H), 2.61 (br s, 1H),1.92-2.02 (m, 2H), 1.81-1.89 (m, 2H), 1.43 (s, 9H), 1.07-1.24 (m, 4H)ppm; ¹³C NMR (D₆-DMSO) δ154.9, 77.3, 49.7, 48.9, 35.1, 31.4, 28.3 ppm;ES-MS (M+1) calcd 215.17, found 215.22.

[0332] General Procedure for Peptide Synthesis. Tat₄₉₋₅₇ (RKKRRQRRR),truncated and alanine-substituted peptides derived from Tat₄₉₋₅₇,Antennapedia₄₃₋₅₈ (RQIKIWFQNRRMKWKK), and homopolymers of arginine(R5-R9) and d-arginine (r5-r9) were prepared with an automated peptidesynthesizer (ABI433) using standard solid-phase Fmoc chemistry (35) withHATU as the peptide coupling reagent. The fluorescein moiety wasattached via a aminohexanoic acid spacer by treating a resin-boundpeptide (1.0 mmol) with fluorescein isothiocyanate (1.0 mmol) and DIEA(5 mmol) in DMF (10 mL) for 12 h. Cleavage from the resin was achievedusing 95:5 TFA/triisopropylsilane. Removal of the solvent in vacuo gavea crude oil which was triturated with cold ether. The crude mixture thusobtained was centrifuged, the ether was removed by decantation, and theresulting orange solid was purified by reverse-phase HPLC (H₂O/CH₃CN in0.1% TFA). The products were isolated by lyophilization andcharacterized by electrospray mass spectrometry. Purity of the peptideswas >95% as determined by analytical reverse-phase HPLC (H₂O/CH₃CN in0.1% TFA).

[0333] All peptides and peptoids synthesized contain an aminohexanoic(ahx) acid moiety attached to the N-terminal amino group with afluorescein moiety (Fl) covalently linked to the amino group of theaminohexanoic acid spacer. The carboxyl terminus of every peptide andpeptoid is a carboxamide.

[0334] Cellular Uptake Assay. The arginine homopolyrners andguanidine-substituted peptoids were each dissolved in PBS buffer (pH7.2) and their concentration was determined by absorption of fluoresceinat 490 nm (ε−67,000). The accuracy of this method for determiningconcentration was established by weighing selected samples anddissolving them in a known amount of PBS buffer. The concentrationsdetermined by UV spectroscopy correlated with the amounts weighed outmanually. Jurkat cells (human T cell line), murine B cells (CH27), orhuman PBL cells were grown in 10% fetal calf serum and DMEM and each ofthese were used for cellular uptake experiments. Varying amounts ofarginine and oligomers of guanidine-substituted peptoids were added toapproximately 3×10⁶ cells in 2% FCS/PBS (combined total of 200 μL) andplaced into microtiter plates (96 well) and incubated for varyingamounts of time at 23° C. or 4° C. The microtiter plates werecentrifuged and the cells were isolated, washed with cold PBS (3×250μL), incubated with 0.05% trypsin/0.53 mM EDTA at 37° C. for 5 min,washed with cold PBS, and resuspended in PBS containing 0.1% propidiumiodide. The cells were analyzed using fluorescent flow cytometry(FACScan, Becton Dickinson) and cells staining with propidium iodidewere excluded from the analysis. The data presented is the meanfluorescent signal for the 5000 cells collected.

[0335] Inhibition of Cellular Uptake with Sodium Azide. The assays wereperformed as previously described with the exception that the cells usedwere preincubated for 30 min with 0.5% sodium azide in 2% FCS/PBS bufferprior to the addition of fluorescent peptides and the cells were washedwith 0.5% sodium azide in PBS buffer. All of the cellular uptake assayswere run in parallel in the presence and absence of sodium azide.

[0336] Cellular Uptake Kinetics Assay. The assays were performed aspreviously described except the cells were incubated for 0.5, 1, 2, and4 min at 4° C. in triplicate in 2% FCS/PBS (50 μl) in microtiter plates(96 well). The reactions were quenched by diluting the samples into 2%FCS/PBS (5 mL). The assays were then worked up and analyzed byfluorescent flow cytometry as previously described.

[0337] Results

[0338] To determine the structural requirements for the cellular uptakeof short arginine-rich peptides, a series of fluorescently-labeledtruncated analogues of Tat₄₉₋₅₇ were synthesized using standardsolid-phase chemistry. See, e.g., Atherton, E. et al. SOLID-PHASEPEPTIDE SYNTHESIS (IRL: Oxford, Engl. 1989). A fluorescein moiety wasattached via an aminohexanoic acid spacer on the amino termini. Theability of these fluorescently labeled peptides to enter Jurkat cellswas then analyzed using fluorescent activated cell sorting (FACS) ( ).The peptide constructs tested were Tat₄₉₋₅₇ (Fl-ahx-RKKRRQRRR): Tat₄₉₋₅₆(Fl-ahx-RKKRRQRR), Ta₄₉₋₅₅ (Fl-ahx-RKKRRQR), Tat₅₀₋₅₇ (Fl-ahx-KKRRQRRR),and Tat₅₁₋₅₇ (Fl-ahx-KRRQRRR). Differentiation between cell surfacebinding and internalization was accomplished throughout by running aparallel set of assays in the presence and absence of sodium azide.Because sodium azide inhibits energy-dependent cellular uptake but notcell surface binding, the difference in fluorescence between the twoassays provided the amount of fluorescence resulting frominternalization.

[0339] Deletion of one arginine residue from either the amine terminus(Tat₅₀₋₅₇) or the carboxyl terminus (Tat₄₉₋₅₆) resulted in an 80% lossof intracellular fluorescence compared to the parent sequence(Tat₄₉₋₅₇). From the one amino acid truncated analogs, further deletionof R-56 from the carboxyl terminus (Tat₄₉₋₅₅) resulted in an additional60% loss of intracellular fluorescence, while deletion of K-50 from theamine terminus (Tat₅₁₋₅₇) did not further diminish the amount ofinternalization. These results indicate that truncated analogs ofTat₄₉₋₅₇ are significantly less effective at the transcellular deliveryof fluorescein into Jurkat cells, and that the arginine residues appearto contribute more to cellular uptake than the lysine residues.

[0340] To determine the contribution of individual amino acid residuesto cellular uptake, analogs containing alanine substitutions at eachsite of Tat₄₉₋₅₇ were synthesized and assayed by FACS analysis (FIG.22). The following constructs were tested: A-49 (Fl-ahx-AKKRRQRRR), A-50(Fl-ahx-RAKRRQRRR), A-51 (Fl-ahx-RKARRQRRR), A-52 (Fl-ahx-RKKARQRRR),A-53 (Fl-ahx-RKKRAQRRR), A-54 (Fl-ahx-RKKRRARRR), A-55(Fl-ahx-RKKRRQARR), A-56 (Fl-ahx-RKKRRQRAR), and A-57(Fl-ahx-RKKRRQRRA). Substitution of the non-charged glutamine residue ofTat₄₉₋₅₇ with alanine (A-54) resulted in a modest decrease in cellularinternalization. On the other hand, alanine substitution of each of thecationic residues individually produced a 70-90% loss of cellularuptake. In these cases, the replacement of lysine (A-50, A-51) orarginine (A-49, A-52, A-55, A-56, A-57) with alanine had similar effectsin reducing uptake.

[0341] To determine whether the chirality of the transporter peptide wasimportant, the corresponding d-(d-Tat₄₉₋₅₇), retro-l-(Tat₅₇₋₄₉), andretro-inverso isomers (d-Tat₅₇₋₄₉) were synthesized and assayed by FACSanalysis (FIG. 23). Importantly, all three analogs were more effectiveat entering Jurkat cells then Tat₄₉₋₅₇. These results indicated that thechirality of the peptide backbone is not crucial for cellular uptake.Interestingly, the retro-l isomer (Tats₅₇₋₄₉) which has three arginineresidues located at the amine terminus instead of one arginine and twolysines found in Tat₄₉₋₅₇ demonstrated enhanced cellular uptake. Thus,residues at the amine terminus appear to be important and that argininesare more effective than lysines for internalization. The improvedcellular uptake of the unnatural d-peptides is most likely due to theirincreased stability to proteolysis in 2% FCS (fetal calf serum) used inthe assays. When serum was excluded, the d- and I-peptides wereequivalent as expected.

[0342] These initial results indicated that arginine content isprimarily responsible for the cellular uptake of Ta₄₉₋₅₇. Furthermore,these results were consistent with our previous results where wedemonstrated that short oligomers of arginine were more effective atentering cells then the corresponding short oligomers of lysine,ornithine, and histidine. What had not been established was whetherarginine homo-oligomers are more effective than Tat₄₉₋₅₇. To addressthis point, Tat₄₉₋₅₇ was compared to the I-arginine (R5-R9) andd-arginine (r5-r9) oligomers. Although Tat₄₉₋₅₇ contains eight cationicresidues, its cellular internalization was between that of R6 and R7(FIG. 24) demonstrating that the presence of six arginine residues isthe most important factor for cellular uptake. Significantly, conjugatescontaining 7-9 arginine residues exhibited better uptake than Tat₄₉₋₅₇.

[0343] To quantitatively compare the ability of these arginine oligomersand Tat₄₉₋₅₇ to enter cells, Michaelis-Menton kinetic analyses wereperformed. The rates of cellular uptake were determined after incubation(3° C.) of the peptides in Jurkat cells for 30, 60, 120, and 240 seconds(Table 1). The resultant K_(m) values revealed that r9 and R9 enteredcells at rates approximately 100-fold and 20-fold faster than Tat₄₇₋₅₉respectively. For comparison, Antermapedia₄₃₋₅₈ was also analyzed andwas shown to enter cells approximately 2-fold faster than Tat₄₇₋₅₉, butsignificantly slower than r9 or R9. TABLE 1 Michaelis-Menton kinetics:Antennapedia₄₃₋₅₈ (Fl-ahx-RQIKIWFQNRRMKWKK). peptide K_(m)(μM) V_(max)Tat₄₉₋₅₇ 770 0.38 Antennapedia₄₃₋₅₈ 427 0.41 R9 44 0.37 r9 7.6 0.38

Example 14 Design and Synthesis of Peptidomimetic Analogs of Tat₄₉₋₅₇

[0344] Methods

[0345] General Procedure for Peptoid Polyamine Synthesis. Peptoids weresynthesized manually using a fritted glass apparatus and positivenitrogen pressure for mixing the resin following the literatureprocedure developed by Zuckermann. See, e.g., Murphy, J. E. et al.,Proc. Natl. Acad. Sci. USA 95, 1517-1522 (1998); Simon, R. J. et al.,Proc. Natl. Acad. Sci. USA 89, 9367-9371 (1992); Zuckermann, R. N. etal., J. Am. Chem. Soc. 114, 10646-10647 (1992). Treatment ofFmoc-substituted Rink amide resin (0.2 mmol) with 20% piperidine/DMF (5mL) for 30 min (2×) gave the free resin-bound amine which was washedwith DMF (3×5 mL). The resin was treated with a solution of bromoaceticacid (2.0 mmol) in DMF (5 mL) for 30 min. This procedure was repeated.The resin was then washed (3×5 mL DMF) and treated with a solution ofmono-Boc diamine (8.0 mmol) in DMF (5 mL) for 12 hrs. These two stepswere repeated until an oligomer of the required length was obtained(Note: the solution of mono-Boc diamine in DMF could be recycled withoutappreciable loss of yield). The resin was then treated withN-Fmoc-aminohexanoic acid (2.0 mmol) and DIC (2.0 mmol) in DMF for 1 hand this was repeated. The Fmoc was then removed by treatment with 20%piperidine/DMF (5 mL) for 30 min. This step was repeated and the resinwas washed with DMF (3×5 mL). The free amine resin was then treated withfluorescein isothiocyanate (0.2 mmol) and DIEA (2.0 mmol) in DMF (5 mL)for 12 hrs. The resin was then washed with DMF (3×5 mL) anddichloromethane (5×5 mL). Cleavage from the resin was achieved using95:5 TFA/triisopropylsilane (8 mL). Removal of the solvent in vacuo gavea crude oil which was triturated with cold ether (20 mL). The crudemixture thus obtained was centrifuged, the ether was removed bydecantation, and the resulting orange solid was purified byreverse-phase HPLC (H₂O/CH₃CN in 0.1% TFA). The products were isolatedby lyophilization and characterized by electrospray mass spectrometryand in selected cases by ¹H NMR spectroscopy.

[0346] General Procedure for Perguanidinylation of Peptoid Polyamines. Asolution of peptoid amine (0.1 mmol) dissolved in deionized water (5 mL)was treated with sodium carbonate (5 equivalents per amine residue) andpyrazole-1-carboxamidine (5 equivalents per amine residue) and heated to50° C. for 24-48 hr. The crude mixture was then acidified with TFA (0.5mL) and directly purified by reverse-phase HPLC (H₂O/CH₃CN in 0.1% TFA).The products were characterized by electrospray mass spectrometry andisolated by lyophilization and further purified by reverse-phase HPLC.The purity of the guanidine-substituted peptoids was >95% as determinedby analytical reverse-phase HPLC (H₂O/CH₃CN in 0.1% TFA).

[0347] Results

[0348] Utilizing the structure-function relationships that had beendetermined for the cellular uptake of Tat₄₇₋₅₉, we designed a set ofpolyguanidine peptoid derivatives that preserve the 1,4 backbone spacingof sidechains of arginine oligomers, but have an oligo-glycine backbonedevoid of stereogenic centers. These peptoids incorporatingarginine-like sidechains on the amide nitrogen were selected because oftheir expected resistance to proteolysis, and potential ease andsignificantly lower cost of synthesis (Simon et al., Proc. Natl. Acad.Sci. USA 89:9367-9371 (1992); Zuckermann, et al., J. Am. Chem. Soc.114:10646-10647 (1992). Furthermore, racemization, frequentlyencountered in peptide synthesis, is not a problem in peptoid synthesis;and the “sub-monomer” peptoid approach allows for facile modification ofside-chain spacers. Although the preparation of an oligurea andpeptoid-peptide hybrid (Hamy, et al, Proc. Natl. Acad. Sci. USA94:3548-3553 (1997)) derivatives of Tat₄₉₋₅₇ have been previouslyreported, their cellular uptake was not explicitly studied.

[0349] The desired peptoids were prepared using the “sub-monomer”approach (Simon et al.; Zuckermann et al.) to peptoids followed byattachment of a fluorescein moiety via an aminohexanoic acid spacer ontothe amine termini. After cleavage from the solid-phase resin, thefluorescently labeled polyamine peptoids thus obtained were converted ingood yields (60-70%) into polyguanidine peptoids by treatment withexcess pyrazole-1-carboxamidine (Bematowicz, et al., J. Org. Chem.57:2497-2502 (1992) and sodium carbonate (as shown in FIG. 25).Previously reported syntheses of peptoids containing isolated N-Argunits have relied on the synthesis of N-Arg monomers (5-7 steps) priorto peptoid synthesis and the use of specialized and expensive guanidineprotecting groups (Pmc, Pbf) (Kruijtzer, et al., Chem. Eur. J.4:1570-1580 (1998); Heizmann, et al. Peptide Res. 7:328-332 (1994). Thecompounds reported here represent the first examples ofpolyguanidinylated peptoids prepared using a perguanidinylation step.This method provides easy access to polyguanidinylated compounds fromthe corresponding polyamines and is especially useful for the synthesisof perguanidinylated homooligomers. Furthermore, it eliminates the useof expensive protecting groups (Pbf, Pmc). An additional example of aperguanidinylation of a peptide substrate using a noveltriflyl-substituted guanylating agent has recently been reported(Feichtinger, et al., J. Org. Chem. 63:8432-8439 (1998)).

[0350] The cellular uptake of fluorescently labeled polyguanidineN-arg5, 7, 9 peptoids was compared to the corresponding d-argininepeptides r5, 7, 9 (similar proteolytic properties) using Jurkat cellsand FACS analysis. The amount of fluorescence measured inside the cellswith N-arg5, 7, 9 was proportional to the number of guanidine residues:N-arg9>N-arg7>N-arg5 (FIG. 26), analogous to that found for r5, 7, 9.Furthermore, the N-arg5, 7, 9 peptoids showed only a slightly loweramount of cellular entry compared to the corresponding peptides, r5, 7,9. The results demonstrate that the hydrogen bonding along the peptidebackbone of Tat₄₉₋₅₇ or arginine oligomers is not a required structuralelement for cellular uptake and oligomeric guanidine-substitutedpeptoids can be utilized in place of arginine-rich peptides as moleculartransporters. The addition of sodium azide inhibited internalizationdemonstrating that the cellular uptake of peptoids was also energydependent.

Example 15 The Effect of Side Chain Length on Cellular Uptake

[0351] After establishing that the N-arg peptoids efficiently crossedcellular membranes, the effect of sidechain length (number ofmethylenes) on cellular uptake was investigated. For a given number ofguanidine residues (5, 7, 9), cellular uptake was proportional tosidechain length. Peptoids with longer sidechains exhibited moreefficient cellular uptake. A nine-mer peptoid analog with asix-methylene spacer between the guanidine head groups and the backbone(N-hxg9) exhibited remarkably higher cellular uptake than thecorresponding d-arginine oligomer (r9). The relative order of uptake wasN-hxg9 (6 methylene)>N-btg9 (4 methylene)>r9 (3 methylene)>N-arg9 (3methylene) >N-etg9 (2 methylene) (FIG. 27). Of note, the N-hxg peptoidsshowed remarkably high cellular uptake, even greater than thecorresponding d-arginine oligomers. The cellular uptake of thecorresponding heptamers and pentamers also showed the same relativetrend. The longer sidechains embodied in the N-hxg peptoids improved thecellular uptake to such an extent that the amount of internalization wascomparable to the corresponding d-arginine oligomer containing one moreguanidine residue (FIG. 28). For example, the N-hxg7 peptoid showedcomparable cellular uptake to r8.

[0352] To address whether the increase in cellular uptake was due to theincreased length of the sidechains or due to their hydrophobic nature, aset of peptoids was synthesized containing cyclohexyl sidechains. Theseare referred to as the N-chg5, 7, 9 peptoids. These contain the samenumber of sidechain carbons as the N-hxg peptoids but possess differentdegrees of freedom. Interestingly, the N-chg peptoid showed much lowercellular uptake activity than all of the previously assayed peptoids,including the N-etg peptoids (FIG. 29). Therefore, the conformationalflexibility and sterically unencumbered nature of the straight chainalkyl spacing groups is important for efficient cellular uptake.

[0353] Discussion

[0354] The nona-peptide, Tat₄₉₋₅₇, has been previously shown toefficiently translocate through plasma membranes. The goal of thisresearch was to determine the structural basis for this effect and usethis information to develop simpler and more effective moleculartransporters. Toward this end, truncated and alanine substitutedderivatives of Tat₄₉₋₅₇ conjugated to a fluoroscein label was prepared.These derivatives exhibited greatly diminished cellular uptake comparedto Tat₄₉₋₅₇, indicating that all of the cationic residues of Tat₄₉₋₅₇are required for efficient cellular uptake. When compared with ourprevious studies on short oligomers of cationic oligomers, thesefindings suggested that an oligomer of arginine might be superior toTat₄₉₋₅₇ and certainly more easily and cost effectively prepared.Comparison of short arginine oligomers with Tat₄₉₋₅₇ showed that membersof the former were indeed more efficiently taken into cells. This wasfurther quantified for the first time bt Michaelis-Menton kineticsanalysis which showed that the R9 and r9 oligomers had Km values 30-foldand 100-fold greater than that found for Tat₄₉₋₅₇.

[0355] Given the importance of the guanidino head group and the apparentinsensitivity of the oligomer chirality revealed in our peptide studies,we designed and synthesized a novel series of polyguanidine peptoids.The peptoids N-arg5, 7, 9, incorporating the arginine sidechain,exhibited comparable cellular uptake to the corresponding d-argininepeptides r5, 7, 9, indicating that the hydrogen bonding along thepeptide backbone and backbone chirality are not essential for cellularuptake. This observation is consistent with molecular models of thesepeptoids, arginine oligomers, and Tat₄₉₋₅₇, all of which have a deeplyembedded backbone and a guanidinium dominated surface. Molecular modelsfurther reveal that these structural characteristics are retained invarying degree in oligomers with different alkyl spacers between thepeptoid backbone and guanidino head groups. Accordingly, a series ofpeptoids incorporating 2-(N-etg), 4-(N-btg), and 6-atom (N-hxg) spacersbetween the backbone and sidechain were prepared and compared forcellular uptake with the N-arg peptoids (3-atom spacers) and d-arginineoligomers. The length of the sidechains had a dramatic affect oncellular entry. The amount of cellular uptake was proportional to thelength of the sidechain with N-hxg>N-btg>N-arg>N-etg. Cellular uptakewas improved when the number of alkyl spacer units between the guanidinehead group and the backbone was increased. Significantly, N-hxg9 wassuperior to r9, the latter being 100-fold better than Tat₄₉₋₅₇. Thisresult led us to prepare peptoid derivatives containing longer octylspacers (N-ocg) between the guanidino groups and the backbone. Issuesrelated to solubility prevented us from testing these compounds.

[0356] Because both perguanidinylated peptides and perguanidinylatedpeptoids efficiently enter cells, the guanidine head group (independentof backbone) is apparently the critical structural determinant ofcellular uptake. However, the presence of several (over six) guanidinemoieties on a molecular scaffold is not sufficient for active transportinto cells as the N-chg peptoids did not efficiently translocate intocells. Thus, in addition to the importance of the guanidine head group,there are structure/conformational requirements that are significant forcellular uptake.

[0357] In summary, this investigation identified a series of structuralcharacteristics including sequence length, amino acid composition, andchirality that influence the ability of Tat₄₉₋₅₇ to enter cells. Thesecharacteristics provided the blueprint for the design of a series ofnovel peptoids, of which 17 members were synthesized and assayed forcellular uptake. Significantly, the N-hxg9 transporter was found to besuperior in cell uptake to r9 which was comparable to N-btg9. Hence,these peptoid transporters proved to be substantially better thanTat₄₉₋₅₇. This research established that the peptide backbone andhydrogen bonding along that backbone are not required for cellularuptake, that the guanidino head group is superior to other cationicsubunits, and most significantly, that an extension of the alkyl chainbetween the backbone and the head group provides superior transporters.In addition to better uptake performance, these novel peptoids offerseveral advantages over Tat₄₉₋₅₇ including cost-effectiveness, ease ofsynthesis of analogs, and protease stability. These features along withtheir significant water solubility (>100 mg/mL) indicate that thesenovel peptoids could serve as effective transporters for the moleculardelivery of drugs, drug candidates, and other agents into cells.

Example 16 Synthesis of Itraconazole-Transporter Conjugate

[0358] This Example provides one application of a general strategy forattaching a delivery-enhancing transporter to a compound that includes atriazole structure. The scheme, using attachment of itraconazole to anarginine (r7) delivery-enhancing transporter as an example, is shown inFIG. 30. In the scheme, R is H or alkyl, n is 1 or 2, and X is ahalogen.

[0359] The reaction involves making use of quaternization of a nitrogenin the triazole ring to attach an acyl group that has a halogen (e.g.,Br, Fl, I) or a methyl ester. Compound 3 was isolated by HPLC. ProtonNMR in D₂O revealed itraconazole and transporter peaks.

[0360] The methyl ester provided yields of 70% and greater, whileyields. obtained using the Br-propionic acid/ester pair were 40-50%. Theacyl derivative is then reacted with the amine of the delivery-enhancingtransporter to form the conjugate. Alternatively, the halogenated acylgroup can first be attached to the transporter molecule through an amidelinkage, after which the reaction with the drug compound is conducted.

Example 17 Preparation of FK506 Conjugates

[0361] This Example describes the preparation of conjugates in whichFK506 is attached to a delivery-enhancing transporter. Two differentlinkers were used, each of which released FK506 at physiological pH (pH5.5 to 7.5), but had longer half-lives at more acidic pH. These schemesare diagrammed in FIGS. 31A and B.

[0362] Linker 1: 6-maleimidocaproic Hydrazide Trifluroacetate (Scheme Iand II)

[0363] A solution of FK506 (1) (0.1 g, 124.4 μmol), 6-maleiimidocaproichydrazide trifluoroacetate (2) (0.126 g, 373.2 μmol) and trifluoroaceticacid (catalytic, 1 μL) in anhydrous methanol (5 mL) was stirred at roomtemperature for 36 h. The reaction was monitored by thin layerchromatography that showed almost complete disappearance of the startingmaterial. [TLC solvent system—dichloromethane (95): methanol (5),R_(f)=0.3]. The reaction mixture was concentrated to dryness anddissolved in ethyl acetate (20 mL). The organic layer was washed withwater and 10% sodium bicarbonate solution and then dried over sodiumsulfate, filtered and concentrated. The residue was purified by columnchromatography using dichloromethane (96): methanol (4) as eluent togive the hydrazone 3 (0.116 g, 92%).

[0364] A solution of the above hydrazone (3) (0.025 g, 24.7 μmol),transporter (1×, Bacar₉CCONH₂.9TFA, Bacar₇CCONH₂.7TFA, BacaCCONH₂,NH₂r₇CCONH₂.8TFA, NH₂R₇CCONH₂.8TFA) and diisopropylethylarnine (1×) inanhydrous dimethylformamide (1 mL) were stirred under nitrogen at roomtemperature for 36h when TLC indicated the complete disappearance of thestarting hydrazone. Solvent was evaporated from the reaction mixture andthe residue purified by reverse phase HPLC using trifluoroacetic acidbuffered water and acetonitrile.

[0365] Yields of conjugates with various transporters:

[0366] Conjugate with Bacar₉CCONH₂.9TFA (4)-73%

[0367] Bacar₇CCONH₂.7TFA (5)-50%

[0368] BacaCCONH₂ (6)-52.9%

[0369] NH₂r₇CCONH₂.8TFA (7)-43.8%

[0370] NH₂R₇CCONH₂.8TFA (8)-62.8%

[0371] Structures of all the products were confirmed by 1 H-NMR spectraand TOF MS analysis.

[0372] Linker 2: 2-(2-pyridinyldithio) Ethyl Hydrazine Carboxylate(Scheme III and IV)

[0373] A solution of FK506 (1) (0.1 g, 124.4 μmol),2-(2-pyridinyldithio) ethyl hydrazine carboxylate (9) (0.091 g, 373.2μmol) and trifluoroacetic acid (catalytic, 1 μL) in anhydrous methanol(5 mL) was stirred at room temperature for 16 h. The reaction wasmonitored by thin layer chromatography that showed almost completedisappearance of the starting material. [TLC solvent system—ethylacetate R_(f)0.5]. The reaction mixture was concentrated to dryness anddissolved in ethyl acetate (20 mL). The organic layer was washed withwater and 10% sodium bicarbonate solution and then dried over sodiumsulfate, filtered and concentrated. The residue was purified by columnchromatography using dichloromethane (97): methanol (3) as eluent togive the hydrazone 10 (0.091 g, 71%)

[0374] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference for all purposes.

What is claimed is:
 1. A method for enhancing delivery of a compoundinto and across one or more layers of an animal epithelial orendothelial tissue, the method comprising: contacting the epithelialtissue with a conjugate that comprises the compound and adelivery-enhancing transporter, wherein the delivery-enhancingtransporter comprises sufficient guanidino or amidino moieties toincrease delivery of the conjugate into and across one or more intactepithelial or endothelial tissue layers compared to delivery of thecompound in the absence of the delivery-enhancing transporter.
 2. Themethod of claim 1, wherein the delivery-enhancing transporter comprisesa non-peptide backbone.
 3. The method of claim 1, wherein thedelivery-enhancing transporter is not attached to an amino acid sequenceto which the delivery enhancing transporter molecule is attached in anaturally occurring protein.
 4. The method of claim 1, wherein thedelivery-enhancing transporter comprises from 5 to 25 guanidino oramidino moieties.
 5. The method of claim 4, wherein thedelivery-enhancing transporter comprises between 7 and 15 guanidinomoieties.
 6. The method of claim 1, wherein the delivery-enhancingtransporter comprises at least 6 contiguous guanidino and/or amidinomoieties.
 7. The method of claim 1, wherein the delivery-enhancingtransporter consists essentially of 5 to 50 amino acids, at least 50percent of which amino acids are arginines or analogs thereof.
 8. Themethod of claim 7, wherein the delivery-enhancing transporter comprises5 to 25 arginine residues or analogs thereof.
 9. The method of claim 8,wherein at least one arginine is a D-arginine.
 10. The method of claim9, wherein all of the arginines are D-arginines.
 11. The method of claim7, wherein at least 70 percent of the amino acids that comprise thedelivery-enhancing transporter are arginines or arginine analogs. 12.The method of claim 7, wherein the delivery-enhancing transportercomprises at least 5 contiguous arginines or arginine analogs.
 13. Themethod of claim 1, wherein the compound is attached to the deliveryenhancing transporter by a linker.
 14. The method of claim 13, whereinthe linker is a releasable linker which releases the compound from thedelivery-enhancing transporter after the compound has passed into andthrough one or more layers of an epithelial or endothelial tissue. 15.The method of claim 14, wherein the compound is biologically active uponrelease from the linker.
 16. The method of claim 1, wherein the compoundis substantially inactive when conjugated to the delivery-enhancingtransporter.
 17. The method of claim 14, wherein the half-life of theconjugate is between 5 minutes and 24 hours upon contact with theepithelial or endothelial tissue.
 18. The method of claim 17, whereinhalf-life of the conjugate is between 30 minutes and 2 hours uponcontact with the epithelial or endothelial tissue.
 19. The method ofclaim 14, wherein the compound is released from the linker bysolvent-mediated cleavage.
 20. The method of claim 13, wherein theconjugate is substantially stable at acidic pH but the compound issubstantially released from the delivery-enhancing transporter atphysiological pH.
 21. The method of claim 13, wherein the conjugate hasa structure selected from the group consisting of structures 3, 4, or 5,as follows:

wherein: R₁—X comprises the compound; X is a functional group on thecompound to which the linker is attached; Y is N or C; R₂ is hydrogen,alkyl, aryl, acyl, or allyl; R₃ comprises the delivery-enhancingtransporter; R₄ is substituted or unsubstituted S, O, N or C; R₅ is OH,SH or NHR₆; R₆ is hydrogen, alkyl, aryl, acyl or allyl; k and m are eachindependently selected from 1 and 2; and n is 1 to
 10. 22. The method ofclaim 21, wherein X is selected from the group consisting of N, O, S,and CR₇R₈, wherein R₇ and R₈ are each independently selected from thegroup consisting of H and alkyl.
 23. The method of claim 21, wherein theconjugate comprises structure 3 and R₂ is selected to obtain a conjugatehalf-life of between 5 minutes and 24 hours.
 24. The method of claim 23,wherein R₂ is selected to obtain a conjugate half-life of between 5minutes and 24 hours.
 25. The method of claim 21, wherein the conjugatecomprises structure 3, Y is N, and R₂ is methyl, ethyl, propyl, butyl,allyl, benzyl or phenyl.
 26. The method of claim 25, wherein R₂ isbenzyl; k, m, and n are each 1, and X is O.
 27. The method of claim 21,wherein the conjugate comprises structure 4; R₄ is S; R₅ is NHR₆; and R₆is hydrogen, methyl, allyl, butyl or phenyl.
 28. The method of claim 21,wherein the conjugate comprises structure 4; R₅ is NHR₆; R₆ is hydrogen,methyl, allyl, butyl or phenyl; and k and m are each
 1. 29. The methodof claim 20, wherein the conjugate comprises structure 6 as follows:

wherein: R₁—X comprises the compound; X is a functional group on thecompound to which the linker is attached; Ar is an aryl group having theattached radicals arranged in an ortho or para configuration, which arylgroup can be substituted or unsubstituted; R₃ comprises thedelivery-enhancing transporter; R₄ is substituted or unsubstituted S, O,N or C; R₅ is OH, SH or NHR₆; R₆ is hydrogen, alkyl, aryl, acyl orallyl; and k and m are each independently selected from 1 and
 2. 30. Themethod of claim 29, wherein X is selected from the group consisting ofN, O, S, and CR₇R₈, wherein R₇ and R₈ are each independently selectedfrom the group consisting of H and alkyl.
 31. The method of claim 29,wherein R₄ is S; R₅ is NHR₆; and R₆ is hydrogen, methyl, allyl, butyl orphenyl.
 32. The method of claim 1, wherein the conjugate comprises atleast two delivery-enhancing transporters.
 33. The method of claim 1,wherein the rate of delivery of the compound into and across the intactepithelial or endothelial tissue layer or layers is increased.
 34. Themethod of claim 1, wherein the amount of compound delivered into andacross the intact epithelial or endothelial tissue layer or layers isincreased.
 35. The method of claim 1, wherein delivery of the compoundinto and across the intact epithelial or endothelial tissue layers issignificantly greater than that of the compound conjugated to the basicHIV tat peptide consisting of residues 49-57.
 36. The method of claim35, wherein delivery of the compound into and across the intactepithelial tissue layers is at least two-fold greater than that of thecompound conjugated to the basic HIV tat peptide consisting of residues49-57.
 37. The method of claim 36, wherein delivery of the conjugateinto and across the intact epithelial tissue layers is at least six-foldgreater than that of the compound conjugated to the basic HIV tatpeptide consisting of residues 49-57.
 38. The method of claim 1, whereinthe compound is a diagnostic imaging or contrast agent
 39. The method ofclaim 1, wherein the compound is a non-nucleic acid.
 40. The method ofclaim 1, wherein the compound is a non-polypeptide.
 41. The method ofclaim 1, wherein the compound exerts its biological effect while orafter passing into both the epidermis and the dermis.
 42. The method ofclaim 41, wherein the compound acts upon immune cells present in thedermis.
 43. The method of claim 1, wherein the compound is a therapeuticfor skin disorders or a cosmetic.
 44. The method of claim 43, whereinthe compound is selected from the group consisting of antibacterials,antifungals, antivirals, antiproliferatives, immunosuppressives,vitamins, analgesics, and hormones.
 45. The method of claim 1, whereinthe epithelial tissue comprises a blood vessel and the compound entersthe blood vessel from the epithelial tissue.
 46. The method of claim 45,wherein the compound exerts its biological effect after entry into thecapillary system.
 47. The method of claim 45, wherein the compound is asystemically active agent.
 48. The method of claim 1, wherein thecompound is selected from the group consisting of antibacterials,antifungals, antivirals, antiproliferatives, hormones,antiinflammatories, vitamins, and analgesics.
 49. The method of claim48, wherein the compound is an antiinflammatory agent.
 50. The method ofclaim 49, wherein the compound is selected from the group consisting ofcorticosteroids, NSIADs, cromolyn, and nedocromil.
 51. The method ofclaim 48, wherein the compound is an antifungal agent.
 52. The method ofclaim 51, wherein the antifungal agent is an azole compound.
 53. Themethod of claim 52, wherein the azole compound is selected from thegroup comprising itraconazole, myconazole and fluconazole.
 54. Themethod of claim 1, wherein the compound is selected from the groupcomprising caffeine, proline, salicylic acid and vitamin E.
 55. Themethod of claim 1, wherein the compound is a therapeutic agent.
 56. Themethod of claim 55, wherein the therapeutic agent is selected from thegroup consisting of cyclosporin, insulin, a vasopressin, a leucineenkephalin, Asu-eel calcitonin, 5-fluorouracil, a salicylamide, aβ-lactone, an ampicillin, a penicillin, a cephalosporin, a β-lactamaseinhibitor, a quinolone, a tetracycline, a macrolide, a gentamicin,acyclovir, ganciclovir, a trifluoropyridine, and pentamidine.
 57. Themethod of claim 1, wherein the epithelial tissue is skin.
 58. The methodof claim 57, wherein the conjugate is applied to intact skin.
 59. Themethod of claim 57, wherein the compound is delivered into and acrossone or more of the stratum corneum, stratum granulosum, stratum lucidumand stratum germinativum.
 60. The method of claim 57, wherein thecompound crosses the stratum corneum in the absence of skinpretreatment.
 61. The method of claim 57, wherein the conjugate isadministered topically and the compound is taken up by cells thatcomprise the follicular or interfollicular epidermis.
 62. The method ofclaim 57, wherein the conjugate is administered by a transdermal patch.63. The method of claim 57, wherein the conjugate is administeredtopically and the compound crosses into and across one or both of thepapillary dermis and the reticular dermis.
 64. The method of claim 63,wherein the compound is taken up by cells present in the dermis.
 65. Themethod of claim 64, wherein the compound is taken up by one or morecells selected from the group consisting of fibroblasts, epithelialcells and immune cells.
 66. The method of claim 1, wherein theepithelial tissue comprises a mucous membrane.
 67. The method of claim66, wherein the conjugate is administered by an oral, nasal, pulmonary,buccal, rectal, transdermal, vaginal or ocular route.
 68. The method ofclaim 66, wherein the compound is a systemically active agent.
 69. Themethod of claim 68, wherein the compound is selected from the groupconsisting of antibacterials, antifungals, antivirals,antiproliferatives, hormones, antiinflammatories, vitamins, andanalgesics.
 70. The method of claim 1, wherein the epithelial tissue isgastrointestinal epithelium.
 71. The method of claim 70, wherein thecompound acts in the gastrointestinal epithelium.
 72. The method ofclaim 70, wherein the compound is a therapeutic for a condition selectedfrom the group consisting of Crohn's disease, ulcerative colitis,gastrointestinal ulcers, Crohn's disease, peptic ulcer disease, andabnormal proliferative diseases.
 73. The method of claim 72, wherein thecompound is a therapeutic for ulcers and is selected from the groupconsisting of an H₂ histamine inhibitor, an inhibitor of theproton-potassium ATPase, and an antibiotic directed at Helicobacterpylori.
 74. The method of claim 1, wherein the epithelial tissue isbronchial epithelium.
 75. The method of claim 74, wherein the compoundis a therapeutic agent for treating a bronchial condition selected fromthe group consisting of cystic fibrosis, asthma, allergic rhinitis, andchronic obstructive pulmonary disease.
 76. The method of claim 75,wherein the condition is asthma and the therapeutic agent is selectedfrom the group consisting of an antiinflammatory agent, abronchodialator, and an immunosuppressive drug.
 77. The method of claim75, wherein the therapeutic agent is an antiinflammatory agent selectedfrom the group consisting of a corticosteroid, cromolyn, and nedocromil.78. The method of claim 74, wherein the conjugate is delivered byinhalation.
 79. The method of claim 1, wherein the endothelial tissue isa blood brain barrier.
 80. The method of claim 79, wherein the compoundis a therapeutic for treating ischemia, Parkinson's disease,schizophrenia, cancer, acquired immune deficiency syndrome (AIDS),infections of the central nervous system, epilepsy, multiple sclerosis,neurodegenerative disease, trauma, depression, Alzheimer's disease,migraine, pain, and a seizure disorder.
 81. A conjugate that comprisesa) a compound to be delivered into and across one or more layers of ananimal epithelial or endothelial tissue, and b) a delivery-enhancingtransporter that comprises 5 to 25 arginine residues; and c) areleasable linker which releases the compound, in biologically activeform, from the delivery-enhancing transporter after the glucocorticoidor ascomycin has passed into and across one or more layers of theepithelial or endothelial tissue.
 82. The conjugate of claim 81, whereinthe delivery-enhancing transporter comprises 7 to 15 arginine residuesor arginine analogs.
 83. The conjugate of claim 81, wherein thedelivery-enhancing transporter consists essentially of 5 to 50 aminoacids, at least 50 percent of which amino acids are arginines orarginine analogs.
 84. The conjugate of claim 81, wherein thedelivery-enhancing transporter comprises at least 5 contiguous argininesor arginine analogs.
 85. The conjugate of claim 81, wherein thehalf-life of the conjugate is between 5 minutes and 24 hours uponcontact with the epithelial or endothelial membrane.
 86. The conjugateof claim 81, wherein the compound is released from the linker bysolvent-mediated cleavage.
 87. The conjugate of claim 81, wherein theconjugate is substantially stable at acidic pH but the compound issubstantially released from the delivery-enhancing transporter atphysiological pH.
 88. The conjugate of claim 81, wherein the conjugateis selected from the group consisting of structures 3, 4, or 5, asfollows:

wherein: R₁—X comprises the compound; X is a functional group on thecompound to which the linker is attached; Y is N or C; R₂ is hydrogen,alkyl, aryl, acyl, or allyl; R₃ comprises the delivery-enhancingtransporter; R₄ is substituted or unsubstituted S, O, N or C; R₅ is OH,SH or NHR₆; R₆ is hydrogen, alkyl, aryl, acyl or allyl; k and m are eachindependently selected from 1 and 2; and n is 1 to
 10. 89. The conjugateof claim 88, wherein X is selected from the group consisting of N, O, S,and CR₇R₈ wherein R₇ and R₈ are each independently selected from thegroup consisting of H and alkyl.
 90. The conjugate of claim 88 whereinthe conjugate comprises structure 3, Y is N, and R₂ is methyl, ethyl,propyl, butyl, allyl, benzyl or phenyl.
 91. The conjugate of claim 90,wherein R₂ is phenyl; k, m, and n are each 1, and X is O.
 92. Theconjugate of claim 88, wherein the linker comprises structure 4; R₄ isS; R₅ is NHR₆; and R₆ is hydrogen, methyl, allyl, butyl or phenyl. 93.The conjugate of claim 88, wherein the conjugate comprises structure 4;R₅ is NHR₆; R₆ is hydrogen, methyl, allyl, butyl or phenyl; and k and mare each
 1. 94. The conjugate of claim 88, wherein the conjugatecomprises:

wherein Ph is phenyl.
 95. The conjugate of claim 86, wherein theconjugate comprises structure 6 as follows:

wherein: R₁—X comprises the compound; X is a functional group on thecompound to which the linker is attached; Ar is an aryl group having theattached radicals arranged in an ortho or para configuration, which arylgroup can be substituted or unsubstituted; R₃ comprises thedelivery-enhancing transporter; R₄ is substituted or unsubstituted S, O,N or C; R₅is OH, SH or NHR₆; R₆ is hydrogen, alkyl, aryl, acyl or allyl;and k and m are each independently selected from 1 and
 2. 96. Theconjugate of claim 95, wherein X is selected from the group consistingof N, O, S, and CR₇R₈, wherein R₇ and R₈ are each independently selectedfrom the group consisting of H and alkyl.
 97. The conjugate of claim 95,where R₄ is S; R₅ is NHR₆; and R₆ is hydrogen, methyl, allyl, butyl orphenyl.
 98. The conjugate of claim 95, wherein the conjugate comprises:


99. A transdermal drug formulation comprising: a therapeuticallyeffective amount of a therapeutic agent; a delivery-enhancing polymerthat comprises sufficient guanidino or amidino sidechain moieties toincrease delivery of the conjugate across one or more layers of ananimal epithelial tissue compared to the trans-epithelial tissuedelivery of the biologically active agent in non-conjugated form; and avehicle suited to transdermal drug administration.
 100. The formulationof claim 99, wherein the formulation is in a topical dosage form. 101.The formulation of claim 100, wherein the topical dosage form is atransdermal patch.